Nucleophosmin protein (NPM) mutants, corresponding gene sequences and uses thereof

ABSTRACT

The invention relates to new nucleophosmin protein (NPM) mutants, corresponding gene sequences and relative uses thereof for diagnosis, monitoring of minimal residual disease; prognostic evaluation and therapy of the acute myeloid leukaemia (AML)

The present invention refers to new nucleophosmin protein mutants,corresponding gene sequences and diagnosis use thereof, monitoring ofthe minimal residual disease, prognostic evaluation and therapy of theacute myeloid leukaemia (LAM).

More particularly, the invention refers to new mutants of thecytoplasmic nucleophosmin protein (NPM) and corresponding gene sequencesand use thereof as markers for the diagnosis, prognosis and therapy ofthe acute myeloid leukaemia with normal karyotype.

The primary acute myeloid leukaemia, the more common leukaemic form inadults, is a disease originated from the bone marrow, more frequentlyfrom a pluripotent or multipotent stem cell, already “committed” for themyelopoiesis. The neoplastic transformation modifies the mechanismsregulating the proliferation and differentiation of the stem cell bypreventing the maturation of its progeny. The consequence of this eventis an accumulation, mainly in the bone marrow and then in the peripheralblood and in other organs and tissues, of leukaemic cells (or blastic)that proliferate autonomously.

To date, LAMs are divided into different prognostic groups based oncytogenetic analyses and molecular biology, in order to program the besttreatment. Currently, the LAM therapy primarily is based on thesequential administration of different active chemotherapeutic drugsagainst the leukaemic cells.

There are different steps in the treatment of LAMs. The first step,named induction therapy, aims to destroy the most part of the leukaemiccells with the objective to lead the patients to the attainment of theso-called complete hematological remission, namely disappearance of theleukaemic cells with normalization of the peripheral and medullaryhematological data. The destruction of the leukaemic cells remainingafter this first step of therapy (so-called minimal residual disease)can be achieved through the continuation of the chemotherapy(maintenance or intensification or re-induction), followed or not byautologous or allogenic transplant (according to the presence or absenceof negative prognostic factors and availability of a donor).Unfortunately, because of the aggressiveness of these malignant tumours,the treatment is decisive only for 30% of the patients. Three are mainusable approaches in order to improve the diagnosis and survival of theLAM affected patients: i) providing rapid and specific diagnostic testsin order to achieve a more accurate diagnosis and be able in monitoringthe so-called “minimal residual disease”; ii) identification ofbiological prognostic factors that allow to stratify the therapeutictreatment according to “risk categories” and iii) development of newmore targeting therapy forms that allow to interfere with the neoplastictransformation mechanisms induced by some genetic lesions.

Some of these objectives have been pursued in some LAM subtypes, asthose with the translocation t (8;21) or inv (16), which can beidentified/monitored with extreme accuracy using cytogenetictechniques/FISH or RT-PCR, and show a prognosis better than other formsof leukaemia. Even, in some LAM subtypes as the promyelocytic leukaemiawith translocation (15; 17), the employment, in combination with thechemotherapy and the trans-retinoic acid or ATRA (an agent that directlyacts on the genetic lesion) has led to an evident improvement in thesurvival of these patients.

Nevertheless these diagnostic/therapeutic improvements concern only aminority of LAMs. In fact a major part of LAM, about 40% of the cases,in the cytogenetic analysis show a normal karyotype (Grimwade et al.,1998) and represents clinically and biologically an heterogeneouscategory (Grimwade et al., 1998; Schnittger et al., 2002; Byrd et al,2002).

The analysis of LAM gene expression with normal karyotype has beenproposed as means for characterizing different prognosis subgroups(Bullinger et al, 2004; Valk et al, 2004), but it has not allowed toidentify genetic lesions specifically associated with the normalkaryotype. Unique genetic lesions till now associated with the normalkaryotype map at level of FLT3 (Schnittger et al., 2002; Frohling etal., 2002), CEBPα (Pabst et al., 2001), and MLL (Steudel ef to the.,2003) genes. Nevertheless, they cannot be considered normalkaryotype-specific because they are present also in cases of LAM withgreater chromosomal translocations (Carnicer et al., 2004) besides inthe acute secondary myeloid leukaemia (Christiansen et al., 2001).

Therefore, up to now, there are not diagnostic/prognostic assays ormolecular markers, which allow to detect and discriminate specificallythe primary normal karyotype LAMs, whose characterization andclassification is still based on fallacious morphological criterions. Inaddition, the ignorance about the genetic lesion represents animpediment for the monitoring of the minimal residual disease (withrelevant difficulty in the therapeutic choices) and to the potentialdevelopment of new forms of molecular therapy for this category of LAM.

From the above it is clear the need to provide new diagnostic andprognostic markers for the primary acute myeloid leukaemia with normalkaryotype, and new molecular targets for the specific therapy of thissubtype of leukaemia.

The authors of the present invention have now identified mutants of thenucleophosmin gene (NPM) and nucleophosmin protein codified from thesame, specifically associated to normal karyotype LAM.

The nucleophosmin (NPM) is a protein largely restricted at the nucleolus(Cordell et al., 1999) that acts as shuttle from the nucleus tocytoplasm (Borer et al., 1989). It is a “chaperon” molecule (Dumbar etal., 1989), probably involved in the prevention of the aggregation ofthe proteins in the nucleolus and regulation of the assemblage andtransport of the pre-ribosomal structures through the nuclear membrane.It is also a target of CDK2/ciclin E in the duplication of thecentrosome (Okuda et al., 2000) and is involved in the regulation of thetumour suppression mechanism mediated by Arf-p53 (Bertwistle et al.,2004; Colombo et al., 2002; Kurki et al., 2004). In the murine model(“knock-out” mouse), NPM gene seems to play a fundamental role in theregulation of the haemopoiesis (Grisendi et al., 2005).

The NPM gene is involved in the chromosomal translocations of leukaemiasand lymphomas resulting in the formation of fusion proteins, such as,for example, NPM-ALK (Morris et al., 1994), NPM RARα (Redner et al,1996), and NPM-MLF1 (Yoneda-Kato et al., 1996), that preserve only theN-terminal region of NPM molecule (Falini et al., 1999; Falini et al.,2002). The nucleophosmin is supposed to contribute to oncogenesis byactivating the oncogenic potential of the fusion partner (ALK, MLF1,RARα (Bischof et al, 1997).

Since NPM is presumed to have a role in the tumour suppression mediatedby Arf/p53, the physiological traffic alterations from the nucleus tothe cytoplasm could be critical during the transformation. Alterationsin the sub-cellular distribution of NPM and/or fusion proteinscontaining NPM can be revealed by immuno-histochemistry studies. Forexample, in the so-called ALK positive lymphomas with t(2;5) (Falini etal., 1999) and acute leukaemia with t(3;5) is observed a cytoplasmicdelocalization of NPM protein (Falini et al., 1999; Falini et al., 2002)with respect to the expected nucleolus location of the same NPM (Cordellet al., 1999). This find is due to the reactivity of the anti-NPMmonoclonal antibody with NPM-ALK fusion protein [the product of thet(2;5)] or NPM-MLF1 [the product of the t(2;5)] or NPM-MLF1 [the productof the t(3;5)] and/or with the NPM protein dislocated in the cytoplasmprobably through formation of heterodimers with NPM-MLF1.

The authors of the present invention have now shown that about a thirdof LAMs in adults at the immunohistochemical examination shows anaberrant cytoplasmic distribution of NPM protein (NPMc+) (normallynucleus-restricted) and that such immunohistochemical find is correlatedto the presence in the leukaemic cells of specific mutations at level ofthe eson 12 of the NPM gene (GenBank NM_(—)002520), in the portionencoding for the C-terminal structure of NPM protein (GenBankNP_(—)002511).

The acute leukaemia expressing NPM protein mutants and correspondinggene sequences, named by the authors LAM NPMc+ (Falini et al., 2005),represents a well-distinct entity that is characterized by widemorphological spectrum, normal karyotype, elevated frequency ofmutations of FLT3 gene (“internal tandem duplication”) and good responseto induction therapy. The mutations of NPM gene and consequentdistribution of the mutated NPM protein in the cytoplasm of leukaemiccells represent the more specific and frequent molecular events till nowfound in normal karyotype LAM. The authors of the present invention havealso shown that NPM mutations represent an excellent marker forprognosis (Schnittger et al., 2005) and monitoring the minimal residualdisease normal karyotype LAMs.

Therefore the authors of the present invention have identified mutantsequences of nucleophosmin protein (NPM) and mutants of NPM geneencoding for them, which advantageously can be employed as: markers inthe preparation of diagnostic kits and prognostic markers and formonitoring minimal residual disease, and as therapeutic targets in theprimary normal karyotype-LAMs.

Therefore, the present invention provides a specific method fordiagnosing, within the heterogeneous category of normal karyotype LAMs,a new subtype, called LAM NPMc+, through immunohistochemical studieswith anti-NPM antibodies (identification of cytoplasmic NPM) and/oranalysis of mutations of NPM gene. This observation has importantdiagnostic implications, because, till now, the normal karyotype LAM wasclassified only based on fallacious morphological criterions (Jaffe etal., 2001). Particularly, the authors have used monoclonal antibodiesagainst epitopes resistant to the fixatives of NPM molecule (Cordell etal., 1999; Falini et al., 2002) that make them applicable to analysis ofroutine biopsy samples fixed and included in paraffin such as, forexample, osteomedullary biopsies or bone marrow clots. Since the NPMmutations are always heterozygote, leukaemic cells contain bothwild-type and mutated NPM protein. These two types of proteins cannot bedistinguish each other using the actual anti-NPM monoclonal antibodies.

More specifically, in order to study the sub-cellular distribution ofNPM mutants without the interference of wild-type NPM protein, theauthors have produced a polyclonal antibody (denominated Sil-C) that isable to recognize specifically NPM mutants.

The cytoplasmic location of NPM (and the mutation of NPM gene) isspecifically associated to the normal karyotype. Therefore, itrepresents an excellent immunohistochemical marker for prognosis, sinceit allows the assignment to the “intermediary risk” LAM category (inwhich are included the patients with normal karyotype) of thoseleukaemic patients that cannot be otherwise classified cytogeneticallybecause of insufficient material, deterioration of the biologicalsample, absence of mitosis, difficulty to interpret surely thekaryotype.

In addition, the immunohistochemical test for the demonstration ofcytoplasmic NPM (NPMc+) (predictive at 100% of mutations of exon-12 ofNPM gene) can be considered as predictive factor for prognosis in normalkaryotype LAMs, by identifying survival differences in the LAM NPMc+cases compared with NPMc−. More specifically, the presence of exon 12mutations of NPM in absence of mutations of FLT3 gene has allowed us toidentify a new group of good prognosis myeloid leukaemias with normalkaryotype (Schnittger et al., 2005).

In addition, the immunohistochemical identification of cytoplasmic NPM(NPMc+) can be considered as predictive factor for a good response tothe induction therapy in the normal karyotype LAMs.

The NPM assays at cytoplasmic level or for mutation at gene level,provided by the authors of the present invention, are highly sensitive,specific, simple, economic and rapid diagnostic tests (48-72 hours toachieve the result) and they use immunohistochemical and biomoleculartechniques well known to those skilled in the art. In addition, suchassays can advantageously be employed for monitoring the minimalresidual disease in a situation (normal karyotype) for which until todayare not available molecular or immunophenotypical markers.

In addition, the results of the study carried out by the authors of thepresent invention suggest the provision of new therapeutic treatments ofLAMs NPMc+ by the use of anti-sense oligonucleotides or small RNAs,which, packaged in lipid particles or virus (Downward J., 2004), areable to interfere with the translation and transcription processes ofthe genes carrying the mutations encoding for mutated NPM proteinsaccording to the invention. Among the possible therapeutic applicationsthere is the employment of intracellular antibodies (“intrabodies”)(Stocks M R, 2005) specifically directed against the C-terminal portionof NPM mutants or the use of small molecules (peptides or like) able toinhibit the specific C-terminal region of NPM mutants.

Among the possible therapeutic applications must be included also thosethat interfere with post-translational changes (acetylation,phosphorylation, ubiquitination, etc.) of NPM molecule (mutated andwild-type) and molecules interacting with them or alterations of routesof the cellular signal specifically associated with the presence ofmutated NPM proteins.

In addition, the administration of the mutant NPM protein or portionsthereof (e.g. peptides) or nucleotide sequences encoding for the proteinor portions thereof for the induction of anti-tumour immunity canadvantageously be employed for preventive or therapeutic use.

Therefore are object of the present invention mutated nucleophosminproteins (NPM) characterized in that they have a cytoplasmic locationand comprise an amino acid sequence mutated at level of at least one ofthe tryptophan residues 288 and/or 290, and/or a signal motif of nuclearexport (NES), present in the C-terminal region of the sequence of thehuman nucleophosmin (NP_(—)002511). In a preferred embodiment of theprotein of the present invention, the mutation interests both twotryptophan 288 and 290 (67.5% of all NPM mutants) or only tryptophan290.

In a preferred embodiment of the protein of the present invention saidsignal motif of nuclear export (NES) comprises an amino acid sequenceYxxxYxxYxY where Y is a hydrophobic amino acid selected from the groupusually consisting of leucine, isoleucine, methionine, valine,phenylalanine, and x can be any amino acid or fragments and variantsthereof. Preferably, the sequence YxxxYxxYxY is selected from the groupconsisting of LxxxVxxVxL (SEQ ID No 1), LxxxLxxVxL (SEQ ID No 2),LxxxFxxVxL (SEQ ID No 3), LxxxMxxVxL (SEQ ID No 4), LxxxCxxVxL (SEQ IDNo 5). More preferably LxxxVxxVxL (SEQ ID No 1) (the most common NESmotif) it is selected from the group consisting of LCLAVEEVSL (SEQ ID No6); LCMAVEEVSL (SEQ ID No 7); LCVAVEEVSL (SEQ ID No 8); LSRAVEEVSL (SEQID No 9); LCTAVEEVSL (SEQ ID No 10); LSQAVEEVSL (SEQ ID No 11);LCHAVEEVSL (SEQ ID No 12); LCRAVEEVSL (SEQ ID No 13); LCRGVEEVSL (SEQ IDNo 14); LCQAVEEVSL (SEQ ID No 15); LCAAVEEVSL (SEQ ID No 16); LCKAVEEVSL(SEQ ID No 17). Preferably, LxxxLxxVxL (SEQ ID No 2) is selected fromthe group consisting of LWQSLAQVSL (SEQ ID No 18); LWQSLEKVSL (SEQ ID No19); LWQSLSKVSL (SEQ ID No 20); LCTFLEEVSL (SEQ ID No 21). Yet morepreferably LxxxFxxVxL (SEQ ID No 3) is selected from the groupconsisting of LWQCFAQVSL (SEQ ID No 22); LWQCFSKVSL (SEQ ID No 23);LWQRFQEVSL (SEQ ID No 24); LWQDFLNRL (SEQ ID No 25). In a preferredembodiment of the present invention LxxxMxxVxL (SEQ ID No 4) isLWQSMEEVSL (SEQ ID No 26) or LWQRMEEVSL (SEQ ID No 27). In anotherpreferred embodiment LxxxCxxVxL (SEQ ID No 5) is LWQCCSQVSL (SEQ ID No28).

Preferably, the C-terminal region of the mutated proteins according tothe invention can include also the VSLRK peptide (SEQ ID No 29) in whichthe L-amino acid represents the last amino acid of NES motifs as abovedefined.

In a preferred embodiment the amino acid sequences of NES motif, asabove defined, can comprise further a D-amino acid upstream of L-aminoacid at the N-terminal end of said NES motif (for instance DLCLAVEEVSLRK(SEQ ID No 30); DLCMAVEEVSLRK (SEQ ID No 31); DLCVAVEEVSLRK (SEQ ID No32); DLCLAVEEVSLRK (SEQ ID No 33); DLWQSLAQVSLRK (SEQ ID No 34);DLWQSLEKVSLRK (SEQ ID No 35).

According to a particular aspect of the present invention the mutatedNPM proteins, as above described, can be fused to a reporter proteinthat in turn can be selected from the group consisting of EGFP,β-galactosidase, luciferase, GFP. The fusion proteins can be prepared bymelting DNA encoding for the aforesaid proteins, commercially available,with the peptide of the present invention and then expressing the soprepared fusion product.

The present invention refers to a mutated or fusion NPM protein as abovedescribed, conjugated with a nanoparticle (i.e. Quantum Dot).

Are further object of the present invention the oligonucleotidesequences encoding for the mutated proteins or fragments and variantsthereof, as above defined. The oligonucleotide sequences according tothe invention can be deoxyribonucleotide or ribonucleotide sequences ortheir complementary sequences.

According to a preferred embodiment of the present invention thedeoxyribonucleotide sequences can include one of the sequences havingthe following deposit numbers of GenBank: AY740634, AY740635, AY740636,AY740637 AY740638, AY740639.

In a particular embodiment of the invention the oligonucleotidesequences can be labelled with an agent selected from the groupconsisting of fluorescent substance, biotin, radioisotope, nanoparticle.The labelling can be also useful for the use of the aforesaidoligonucleotide sequences as markers for in vitro diagnosis and/ormonitoring the minimal residual disease and/or prognosis of LAMscharacterized by normal karyotype. Particularly, the marker consists inat least a couple of primers, that can be labelled or not in one or inboth the primers with an agent selected from the group consisting offluorescent substance, biotin, radioisotope, nanoparticle (Quantumdot).In another embodiment the marker consists in at least an oligonucleotideprobe that can be labelled with an agent selected from the groupconsisting of fluorescent substance, biotin, radioisotope, nanoparticle.

Further objects of the present invention are the primers having thefollowing sequences:

i) NPM1_25F: (SEQ ID No 36) 5′-GGTTGTTCTCTGGAGCAGCGTTC-3′; NPM1_111 2R:(SEQ ID No 37) 5′-CCTGGACAACATTTATCAAACACGGTA-3′; ii) NPM1_390F: (SEQ IDNo 38) 5′-GGTCTTAAGGTTGAAGTGTGGT-3′; NPM1_1043_R; (SEQ ID No 39)5′-TCAACTGTTACAGAAATGAAATAAGACG-3′; iii) NPM_940F_mutA (SEQ ID No 40)5′-GAGGCTATTCAAGATCTCTGTCT-3′; NPM1_1112 R (SEQ ID No 41)5′-CCTGGACAACATTTATCAAAGACGGTA-3′; iv) NPM_940F_mutB (SEQ ID No 42)5′-GAGGCTATTCAAGATCTCTGCAT 3′; NPM1_1112 R (SEQ ID No 43)5′-CCTGGACAACATTTATCAAACACGGTA3′; v) NPM_940F_mutC (SEQ ID No 44)5′-GAGGCTATTCAAGATCTCTGCGT-3′; NPM1_1112 R (SEQ ID No 45)5′-CCTGGACAACATTTATCAAACACGGTA-3′; vi) NPM_940F_mutD (SEQ ID No 46)5′-GAGGCTATTCMGATCTCTGCCT-3′; NPM1_1112 R (SEQ ID No 47)5′-CGTGGACAACATTTATCAAACACGGTA-3′; vii) NPM1 -F: (SEQ ID No 48)5′-TTAACTCTCTGGTGGTAGAATGAA-3′; NPM1-R: (SEQ ID No 49)5′-CCAGACTATTTGCCATTCCTAAC-3′; viii) NPM1_89F_BamHI: (SEQ ID No 50)5′-GCCACGGATCCGAAGATTCGATGGAC-3′; NPM1_1044R_EcoRI: (SEQ ID No 51)5′ATCAAGAATTCCAGAAATGAAATAAGA CG-3′; ix) cNPM-F: (SEQ ID No 52)5′-GAAGAATTGCTTCCGGATGACT-3′ and cNPM mut.A-R: (SEQ ID No 53)5′-CTTCCTCCACTGCCAGACAGA-3′ or cNPM mut.B-R: (SEQ ID No 54)5′-TTCCTCCACTGCCATGCAG-3′.

Particularly, the primers i) can be employed for the amplification,preferably by RT-PCR, of the encoding region of NPM gene (NM_(—)002520).The primers ii) can be used alone for the amplification that produces aconstruct with a pCRII-mouse vector (Invitrogen). The primers iii), iv),v), vi) above mentioned have been designed respectively for theamplification by RT-PCR of oligonucleotide sequences encoding for themutated proteins:

A) DLCLAVEEVSLRK (SEQ ID No 30) - primer iii); B) DLCMAVEEVSLRK (SEQ IDNo 31) - primer iv); C) DLCVAVEEVSLRK (SEQ ID No 32) - primer v); D)DLCLAVEEVSLRK (SEQ ID No 33) - primer vi);characterized by having as mutation a duplication of tetranucleotide.Finally, the primers vii) are employed for the amplification by genomicDNA of the exon 12 of the C-terminal region of NPM protein; while theprimers viii) are used for the insertion in an expression vector of thesequences according to the invention.

It is a further object of the present invention a plasmid comprising oneof the above described oligonucleotide sequences. Preferably, saidplasmid can be selected from the group consisting of pGEM-T and pGEM-TEasy (Promega).

A further object of the present invention is represented by anexpression vector (such as, for instance, a viral vector or pEGFP-c1)comprising one of the above described oligonucleotide sequences. In apreferred embodiment of the present invention said vector is pEGFP-c1,that comprises the sequence of EGFP reporter gene.

The present invention also refers to a host cell able to express atleast one of the proteins mutated as above described, said cellcomprising the plasmid with above defined characteristics. Preferably,the host cell is prokaryotic.

In addition it is object of the present invention a cellular line,preferably mammal (yet more preferably murine such as NIH 3T3 and BaF3),comprising the expression vector to be employed as experimental studymodel of LAM NPMc+ or as screening model to test new molecules to beused as potential drugs for such type of leukaemia.

It is a further object of the present invention a method for thepreparation of mutated nucleophosmin proteins (NPM) comprising thefollowing steps of:

a) amplification of the oligonucleotide sequence according to theinvention by RT-PCR through at least a couple of primers;b) introduction of the oligonucleotide sequence amplified in step a) inthe pGEM-T Easy vector and transfer in an expression vector byrestriction enzymes;c) transfection of the expression vector in a mammalian competentcellular line selected among the murine cellular lines NIH 3T3 and BaF3;d) extraction and purification of the mutated proteins.

In a preferred form of the method according to the invention said atleast couple of primers of the step a) is:

NPM1_89F_BamHI: (SEQ ID No 50) 5′-GCCACGGATCCGAAGATTCGATGGAC-3′NPM1_1044R_EcoRI: (SEQ ID No 51) 5′-ATCAAGAATTCCAGAAATGAAATAAGACG-3′.

According to a further aspect the present invention refers to amonoclonal or polyclonal antibody or a fragment thereof (such as, forinstance, a scFv fragment) able to recognize and join selectively atleast an antigenic epitope of the C-terminal region (that can compriseVSLRK) of mutated NPM proteins according to the invention.

According to a particular aspect of the present invention the monoclonaland polyclonal antibodies can be conjugated with an agent selected fromthe group consisting of fluorescent substance, enzyme, radioisotope,nanoparticle, medicine.

Particularly the monoclonal antibodies and the polyclonal antibodiesaccording to the invention are specifically directed against theleukaemia-specific epitopes of the C-terminal of NPM that can comprisethe peptide VSLRK, and/or the mutation of at least one of tryptophanresidues 288 and 290 (preferably of both tryptophan residuescontemporarily or only tryptophan residue 290), and/or the NES motif asabove defined.

It is a further object of the present invention the use of monoclonal orpolyclonal antibodies as markers for in vitro diagnosis and prognosis,for monitoring minimal residual disease and/or preparation of a drug forthe therapy of normal karyotype LAMs. In a preferred embodiment, thepresent invention contemplates the use of intracellular antibodies(“intrabodies”) directed specifically against epitopes of the C-terminalpart of the mutants as medicine to inhibit the activity of mutated NPMproteins.

It is an object of the present invention a diagnostic kit for thedetection of normal karyotype LAMs starting from a biological sample,comprising the antibody or a fragment thereof. Said antibody or fragmentcan be directed against epitopes resistant to the fixatives of thenative NPM protein or against the C-terminal mutated portion of NPM.Said detection can be achieved by immunohistochemical, immunoenzymatic,immunoblotting, immunoprecipitation assays or a combination thereof.

Further object of the present invention is represented by the use of theoligonucleotide sequences or of the nucleophosmin proteins encoded bythem according to the invention, for the preparation of means for invitro diagnosis or prognosis of normal karyotype LAMs and/or monitoringminimal residual disease. Such means for diagnosis are preferablyselected from the group consisting of oligonucleotide probes, primers,epitopes, antibodies.

A particular object of the present invention is represented by theleukaemia-specific antigenic epitope that is created at level of theC-terminal region of mutated nucleophosmin proteins, characterized bycomprising the VSLRK peptide, and/or the mutation of at least one oftryptophan residues 288 and 290 (preferably of both the tryptophanresidues contemporarily or only of the tryptophan residue 290), and/orthe NES motif as above defined.

Further object of the present invention is constituted by a diagnostickit for the detection of the normal karyotype LAMs in a biologicalsample and/or for monitoring minimal residual disease, comprising atleast one of the oligonucleotide sequences according to the invention orportion thereof. Said detection can occur through PCR, RT-PCR, Real-timeor in situ hybridization, Reverse Dot Blot (RBD), Multiple Tissue Array(My) techniques or a combination thereof.

Particularly, the invention refers to a Real-Time PCR diagnostic kit formonitoring minimal residual disease comprising the following components:

i) the couple of primers (SEQ ID No 52) cNPM-F:5′-GAAGAATTGCTTCCGGATGACT-3′ and (SEQ ID No 53) cNPM mut.A-R:5′-CTTCCTCCACTGCCAGACAGA-3′ or (SEQ ID No 54) cNPM mut.B-R:5′-TTCCTCCACTGCCATGCAG-3′ ii) and the probe (SEQ ID No 55)5′FAM-ACCAAGAGGCTATTCAA-MGB-3′.

In addition it is object of the present invention a solid support, suchas for example a membrane, or an array, comprising at least one of theoligonucleotide sequences according to the invention.

According to a further aspect, the present invention refers to a invitro method for the detection of the oligonucleotide sequences encodingfor above described mutated nucleophosmin proteins comprising thefollowing steps of:

a) extraction of RNA from the biological test sample;b) retro-transcription and amplification by RT-PCR or Real-Time PCR bythe use of NPM sequence specific primers (Gen Bank NM_(—)002520) asabove defined;c) purification and sequencing of PCR products by using primers.

In a preferred embodiment of the aforesaid method the primers employedin the step b) are the couples of forward and reverse primers from i) toix) above listed. Preferably, the primers of the step c) are selectedfrom the group consisting of:

(SEQ ID No 36) NPM1_25F: 5′-GGTTGTTCTCTGGAGCAGCGTTC-3′; (SEQ ID No 37)NPM1_1112R: 5′-CCTGGACAACATTTATCAAACACGGTA-3′; (SEQ ID No 38) NPM1_390F:5′-GGTCTTAAGGTTGAAGTGTGGT-3′; (SEQ ID No 39) NPM1_1043_R;5′-TCAACTGTTACAGAAATGMATAAGACG-3′.

The same primers i) taken singly as also the primers ii) can be used inthe step c) of the aforesaid method (for the sequencing).

Particularly, in the case in which it is employed the Real-time for thedetection, the “Hybridization probe” or “SYBR green detection” or“Hydrolysis probe” system can alternatively be employed.

In a preferred embodiment the present invention refers to a method thatin the step b) employs a mutation-specific Real Time PCR system. Theprimers used are

cNPM-F: 5′-GAAGAATTGCTTCCGGATGACT-3′ (SEQ ID No 52) andcNPM mut.A-R: 5′-CTTCCTCCACTGCCAGACAGA-3′ SEQ ID No 53) orcNPM mut.B-R: 5′-TTCCTCCACTGCCATGCAG-3′ (SEQ ID No 54)and the probe is 5′FAM-ACCAAGAGGCTATTCAA-MGB-3′ (SEQ ID No 55)as shown in FIG. 13 (see also Example 5).

Further the invention refers to an oligonucleotide probe having thefollowing oligonucleotide sequence 5′FAM-ACCAAGAGGCTATTCAA-MGB-3′ (SEQID No 55).

It is a further object of the present invention a method for thedetection of the oligonucleotide sequences encoding for the abovedescribed mutated nucleophosmin proteins comprising the following stepsof:

a) extraction of RNA from the biological test sample;b) amplification by the use of specific primers for the NPM sequence of(Gen Bank NM_(—)002520);c) detection of the PCR products.

Preferably, the amplification occurs through diagnostic PCR or Real-timePCR. Particularly in the case in which Real-time is employed for thedetection, the “Hybridization probe” or “SYBR green detection” or“Hydrolisis probe” system can alternatively be employed.

In a preferred embodiment of the aforesaid method the primers employedin the step b) are selected from the group consisting of:

i) NPM_940F_mutA: (SEQ ID No 40) 5′-GAGGCTATTCAAGATCTCTGTCT-3′; NPM1_1112R: (SEQ ID No 41) 5′-CCTGGACAACATTTATCAAACACGGTA-3′; or NPM1_390F: (SEQID No 42) 5′-GGTCTTAAGGTTGAAGTGTGGT-3′; NPM1_1043_R; (SEQ ID No 43)5′-TCAACTGTTACAGAAATGAAATAAGACG-3′; ii) NPM_940F_mutB: (SEQ ID No 44)5′-GAGGCTATTCAAGATCTCTGCAT 3′; NPM1_1112R: (SEQ ID No 45)5′-CCTGGACAACATTTATCAAACACGGTA 3′; iii) NPM_940F_mutC (SEQ ID No 46)5′-GAGGCTATTCAAGATCTCTGCGT-3; NPM1_1112R (SEQ ID No 47)5′-CCTGGACAACATTTATCAAACACGGTA-3′; iv) NPM_940F_mutD (SEQ ID No 48)5′-GAGGCTATTCAAGATCTCTGCCT-3′; NPM1_1112 R (SEQ ID No 49)5′-CCTGGACAACATTTATCAAACACGGTA-3′.

Particularly in the case in which Real-time is employed for thedetection the “Hybridization probe” or “SYBR green detection” or“Hydrolisis probe” system can alternatively be employed.

According to a further aspect, the present invention has as object an invitro method for the detection of the cytoplasmic location of NPM in abiological sample comprising the following steps of:

a) contacting the biological sample with at least an antibody or afragment thereof able to detect the cytoplasmic location of NPM;b) detection of the antibody-antigenic epitope bond, by standardimmunofluorescence or immunoenzymatic techniques.

Said biological sample of step a) of the aforesaid method is selectedfrom the group consisting of: paraffin embedded tissue sections,preferably of bone marrow (osteomedullary biopsy or haematic clot) ormedullary smears and blood or liquor cytological samples.

Preferably said antibody of the step a) of the method is a monoclonalantibody. Particularly, can be used monoclonal antibodies directedagainst epitopes resistant to fixatives of NPM (Cordell et al., 1999;Falini et al., 2002) that are able to detect the cytoplasmic location ofthe protein.

In a preferred embodiment the aforesaid method can subsequently includeanother step c) for the detection of mutated NPM proteins in the abovedescribed pathological samples (sections, smears, cytological samples)through antibodies able to recognize and join selectively at least anantigenic epitope of mutated NPM proteins according to the invention orc1) for the detection of oligonucleotide sequences mutated, for example,by amplification methods with specific primers. The primers of the stepc1) can be:

(SEQ ID No 40) NPM_940F_mutA 5′-GAGGCTATTCAAGATCTCTGTCT-3′; (SEQ ID No41) NPM1_1112R 5′-CCTGGACAACATTTATCAAACACGGTA-3′; (SEQ ID No 42)NPM_940F_mutB 5′-GAGGCTATTCAAGATCTCTGCAT 3′; (SEQ ID No 43) NPM1_1112R5′-CCTGGACAACATTTATCAAACACGGTA 3′; (SEQ ID No 44) NPM_940F_mutC5′-GAGGCTATTCAAGATCTCTGCGT-3′; (SEQ ID No 45) NPM1_1112R5′-GCTGGACAACATTTATCAAACACGGTA-3′; (SEQ ID No 46) NPM_940F_mutD5′-GAGGCTATTCAAGATGTCTGCCT-3′; (SEQ ID No 47) NPM1_1112R5′-CCTGGACAACATTTATCAAACACGGTA-3′;

for the identification of the mutations A-D.

In a preferred embodiment of the method according to the invention theantigenic epitope of step c) can comprise the VSLRK peptide, and/or themutation of at least one of tryptophan residues 288 and 290 (preferablyboth tryptophan residues contemporarily or only tryptophan residue 290),and/or the NES motif as above defined.

Further object of the present invention is represented by anti-senseoligonucleotides able of hybridize to at least one of theoligonucleotide sequences encoding for mutated nucleophosmin proteinsaccording to the invention and to interfere with their transcriptionprocess. In a preferred embodiment, it is an object of the presentinvention the use of the anti-sense oligonucleotides for the preparationof a drug for the treatment of primary LAMs.

The present invention has as further object, a ribonucleotide sequence(RNAi) able to hybridize selectively to at least one of theoligonucleotide sequences encoding for mutated nucleophosmin proteinsaccording to the invention and to interfere with their expression. RNAiis a double strand RNA molecule, comprising a combination of sense andanti-sense oligonucleotide strands, which prevents the translation ofthe target mRNAs.

According to a particular aspect, the present invention refers to theuse of the ribonucleotide sequence RNAi, also in the form of microRNA,for the preparation of a drug for the treatment of primary LAMs.

In addition among the possible therapeutic applications, arecontemplated, according to the invention, the molecules interfering withthe post-translational change processes (acetylation, phosphorylation,ubiquitination) of NPM molecule (mutated and wild-type) or withalterations of routes of the cellular signal specifically associatedwith the presence of one of mutated NPM proteins that are object of thepresent invention. In addition, the present invention suggests the useof specific inhibitors of the C-terminal portion of mutant NPM proteinsby small molecules (peptides or others) identified using “SAR by NMR”(Shuker et al., 1996) or other methods.

It is a further object of the present invention the use ofoligonucleotide sequences or nucleophosmin proteins encoded by themaccording to the invention, or portions (peptides) thereof, or acombination thereof, for the preparation of an anti-tumour vaccine. Infact, the nucleophosmin proteins or peptides are usable as vaccinesagainst LAM NPMc+ leukaemias.

Further object of the present invention is represented by apharmaceutical composition comprising at least one of theoligonucleotide sequences or nucleophosmin proteins encoded by themaccording to the invention, or portions (peptides) thereof, along withpharmacologically acceptable excipients and adjuvants.

It is a further object of the present invention an anti-tumour vaccinecomprising at least one of the oligonucleotide sequences ornucleophosmin proteins encoded by them according to the invention, orportions (peptides) thereof, along with pharmacologically acceptableexcipients and adjuvants. Said anti-tumour vaccines are specific for thepreventive and therapeutic treatment of LAM NPMc+.

The present invention finally has also as object a non human transgenicanimal comprising a oligonucleotide sequence encoding for a mutatednucleophosmin protein as above described. Said non human transgenicanimal can be employed as experimental study model for the study of newdrugs for the treatment of LAM NPMc+.

The present invention now will be described by illustrative not limitingway, according to its preferred embodiments, with particular referenceto figures of enclosed drawings, in which:

FIG. 1A shows the histograms relating to the sub-cellular nucleophosminexpression in LAM, respectively, NPMc+ and NPMC− in the left and rightpanel, respectively; in the left panel the arrows indicate leukaemiccells while the arrowhead indicates a residual haemopoietic cell withexpected nucleus-restricted NPM positivity (×1,000); the arrows in theright panel indicate a mitotic cell with expected NPM cytoplasmicexpression (×1,000);

FIG. 1B shows the cytoplasmic NPM expression in 1706 human tumours,which is specific for primary LAMs, not being detectable in otherhaemopietic and extra-haemopietic tumours such as secondary LAMs; acutelymphoid leukaemia (ALL); chronic myeloid leukaemia (CML);myelodysplastic syndromes (MDS); non-Hodgkin lymphomas (NHL) andextra-haemopoietic solid tumours (EXTRA-HEMOP);

FIG. 1C shows the histograms relating to the correlation between NPMsub-cellular expression and morphology in 591 primary LAMs ofGIMEMA/EORTC study plus 70 LAMs of M3 subtype with t(15;17); theLAM-NPMc+s can belong to all FAB subgroups, but are more often M4-M5type, the FAB-M5b form almost always contains NPM mutations.

FIG. 1D shows cytoplasmic NPM expression in erythroid and myeloidleukaemic cell lineage (“multilineage involvement”); in the left panelit is shown the marrow infiltrated by myeloid (arrowhead) and erythroidblasts (arrow); in the central panel are indicated abnormal erythroidprecursors (arrow) and clusters of myeloid blasts (double arrows), bothexpressing NPM at cytoplasmic and nuclear level; arrowhead indicates aresidual haemopoietic cell with expected nucleus-restricted NPM; in theright panel are indicated the cells with the expected nucleus-restrictednucleolin (C23) expression; arrows in middle and right panels indicateleukaemic cells brown double stained for glycophorin (surface) and bluestained for NPM (cytoplasm plus nucleus) or for C23(nucleus-restricted); all figures have been obtained using APAAPimmunoenzymatic technique; ×1,000;

FIG. 2 shows the histograms relating to the association betweencytoplasmic NPM expression and the presence of CD34: FIGS. 2A-C showthat in most cases the cytoplasmic NPM expression associates withCD34-negativity, while NPMc− LAM cases show an opposite behavior; FIG.2D shows a NPMc+ normal karyotype LAM and absence of CD34 expression(osteomedullary biopsy, APAAP technique ×1,000); FIG. 2E shows aNPMc-LAM with karyotype normal and intense CD34 positivity(osteomedullary biopsy, APAAP technique ×1,000); figure F representsFACS analysis of a NPMc+ LAM; left panel: control; central panel: CD34(FITC) and CD133 (PE) lacking leukaemic cells; right panel: CD33 (FITC)and CD13 (PE) co-expressing leukaemic cells;

FIG. 3A refers to analysis of 493 patients with LAM of the GIMEMA/EORTCstudy and shows that the NPMc+ cases associate exclusively to the normalkaryotype (NK: normal karyotype; AK: abnormal karyotype);

FIG. 3B shows analysis of 493 LAM GIMEMA/EORTC study plus 109 patientsnot included in the study (70 acute promyelocytic leukaemias witht(15;17) and 39 LAMs with major chromosomal rearrangements); 24 NPMc+(column “other”) don't associate never to major chromosomalabnormalities and show only smaller abnormalities; none of the NPMc+LAMs associates to major chromosomal abnormalities; 24 NPMc+ cases(column “other”) show only smaller abnormalities;

FIGS. 3C and 3D show the expected NPM localization at nuclear level inNPMc+ LAM of M3 and M4eo type (APAAP technique; ×1,000);

FIG. 3E shows that, in context of normal karyotype LAMs (NK), FLT3-ITDmutations (internal tandem duplication-ITD) occur more frequently in theNPMc+ LAMs compared with NPMc− LAMs (U=not mutated; M=mutated);

FIG. 3F shows that, in context of normal karyotype LAMs (NK), NPMc+forms occur more frequently in more advanced age ranges;

FIG. 3G shows multivariate logistic regression model that establishesthe independent association between cytoplasmic NPM and FLT3-ITD; IndipVar: independent variable; GL: degree of freedom; Sig: significance; OR:risk measurements; *: Normal vs abnormal karyotype (excluded greatergenetic events); M: mutated; U: not mutated;

FIG. 4A is a schematic representation of NPM gene; the dark blocksindicate encoding sequences; not colored blocks indicate 3′- and5′-UTRs; MB is the metal binding domain; Ac: is the acid domain; NLSrepresents nuclear localization signal; NAB represents nucleic acidbinding domain; dark arrows indicate the position on the map of primersfor genomic DNA and not colored arrows indicate primers for cDNAamplification;

FIG. 4B shows wild-type NPM sequence (nucleotides 952-989) aligned withsix mutant variants, named A to F; the capital letters indicatenucleotide insertions; the mutated protein sequence with C-terminalunderlined tryptophan residues (W) is represented on the right, whilethe novel amino acid sequence common to all mutated proteins isindicated in the box area; for each variant, the total number ofaffected cases is given;

FIG. 4C shows sequencing of NPM from one patient hearing mutation A, asobtained by direct sequencing (on top) and after cloning and sequencingof the two variant alleles (middle, wild-type, bottom, mutated allele);the arrows indicate the position wherein allele diverges;

FIG. 4D shows the tridimensional reconstruction at confocal microscopyof NIH 3T3 cells transfected with plasmids encoding for EGFP-taggedwild-type (on top) and mutant NPM alleles (in the bottom); the wild-typeprotein localizes in the nucleoli and nuclear membrane, while themutated NPM shows aberrant cytoplasmic localization;

FIG. 5A shows Western Blot (antibodies Sil-C vs 376) of whole lysedcells of NPMc+ or NPMc− leukaemic cells; Sil-C antibody specificallyrecognizes mutated NPM protein only in LAM cases with NPM mutation(NPMc+);

FIG. 5B shows Western Blot (Sil-C antibody) of a nuclear (N) andcytoplasmic (C) fraction in a leukaemic patient with the mutation A ofNPM: Sil-C recognizes specifically mutated NPM protein in cytoplasmicfraction of NPMc+ primary leukaemic cells; results represent 3independent experiments;

FIGS. 5C-5E show the osteomedullary biopsies of NPMc+ leukaemic cellswith the mutation A: (5C) typical image of FAB-M5 acute myeloidleukaemia; the arrow indicates a big leukaemic cell with kidney-shapednucleus; (5D) Sil-C antibody specifically recognizing the mutant (NPMm),shows a cytoplasm-restricted positivity (arrow); T indicates a bonytrabecula; (5E) the anti-NPM 376 monoclonal antibody recognizes bothwild-type NPM and mutated NPM protein and it stains the leukaemic cellsin the cytoplasm and in nucleus (arrow);

FIGS. 5F-5G show osteomedullary biopsies of NPM mutations lackingpromyelocytic leukaemia (NPMc−): (5F) typical morphology of a LAM-M3(haematoxylin-eosin, ×400); (5G) Sil-C antibody doesn't stain M3leukaemic cells and stains only vessel wall by cross-reactivity; theasterisk indicates the vessel lumen (APAAP technique; counterstaining inhaematoxylin; ×400);

FIG. 6 shows the changes in 288 and 290 tryptophan and the creation of aNES motif in 40 mutant NPM proteins identified in leukaemic patients;Cit: cytoplasmic; nd: unavailable; IH: immunohistochemistry; amino acidsin the panels: NES motif; underlined amino acids: tryptophan residues;

FIG. 7A-B shows HI299 cells transfected with c-DNA encoding for NPMwtand NPM mutants from A to D in the absence (7A) and in the presence (7B)of Leptomycin B (LMB). eGFP-NPMwt is localized in nucleoli both in thepresence and in the absence of LMB. In the absence of LMB, all themutants associated with GFPs (from A to D) show the aberrant cytoplasmiclocalization (7A) while the same mutants associated with eGFP arecompletely replaced in the nucleus in the presence of LMB (7B);

FIG. 7C-E shows the analysis at various time intervals of LMB effects onmutant A joined to eGFP (eGFP-NPMmutA) in N1H-3T3 cells: (7C) images atconfocal microscopy of NIH-3T3 cells at the indicated times where thewhite circles represent the regions (regions of interest, ROI) in whichthe fluorescence intensity of eGFP-NPMmutA has been calculated; (7D)fluorescence measurement in selected areas (ROI) of NIH-3T3 cells; (7E)Western Blot analysis of sub-cellular distribution of eGFP-NPMmutA inNIH-3T3 cells using anti-GFP monoclonal antibody;

FIGS. 8 A-B show confocal microscopy experiments in order to verify there-localization of NPM mutants in the nucleoplasm with Leptomicin B(LMB): (8A) confocal microscopy of NIH-3T3 cells transfected withGFP-mutA or with GFP-NPMwt in the presence or in the absence of LMB;under basal conditions, the mutant is localized only in cytoplasm (ontop in the left) but it repositions in nucleoplasm (on top in the right)after incubation with LMB; GFP-NPMwt localizes in the nucleoli both inthe presence and in the absence of LMB (middle panel, left and right);the lower panel shows cells treated with LMB and transfected withGFP-mutA and stained in nucleoli with anti-nucleolin monoclonal antibody(C23) (arrow); the mutant NPM protein (green) localizes in thenucleoplasm while the nucleolin/C23 (red) is restricted at nucleoli, thenuclear membrane is stained in blue; (8B) confocal microscopy of bonemarrow of a NPMc+ leukaemia with mutation A of NPM (superior panel); theleukaemic cells show myeloid differentiation in medullary aspirate (ontop in the left, arrow) and bony biopsy (on top in the middle); theleukaemic cells, mostly near to a bony trabecula (T), show cytoplasmic(and nuclear) NPM expression (on top in the right); in the middle andinferior panels purified leukaemic cells stained with anti-NPM 376monoclonal antibody and observed at confocal microscopy in the absence(middle panel) and in the presence (inferior panel) of leptomycin B areshown; the images have been reconstructed in 3-D and an electronic cuton the nuclear surface has been carried out in order to better observeNPM localization. Under basal conditions the 376 monoclonal antibodythat doesn't distinguish between NPM wild-type and mutant A, labels boththe nucleoli and the cytoplasm (middle panel); after treatment withleptomycin B NPM is in nucleus again (inferior panel);

FIG. 9A shows the nuclear re-localization of mutant NPM by leptomycin Bin OCI/AML3 cells: (9A) in the absence of LMB, the anti-NPM 376 antibodyunderlines both the nucleoli and the cytoplasm (left); the incubationwith LMB determines a re-localization of NPM in the nucleoplasm (right);

FIG. 9B shows the immunoprecipitation (IP) of lysed cells of OCI-AML3cells and U937 cells with control IgG or anti-NPMm rabbit antibodies(Sil-C) and anti-NPM murine antibody: Western blots with anti-Crm1antibodies are shown in superior panels while those with anti-NPMm(Sil-C) and anti-NPM (CI-376) antibodies are shown in middle andinferior panels; WCL indicates OCI-AML3 lysed cells included as control;the figure shows the physical interaction between mutated NPM proteinand Crm1;

FIG. 10 shows confocal microscope analysis of N1H-3T3 cells transfectedwith wild type eGFP-NPM or mutant eGFP-NPM and shows that NES andtryptophan mutations are both necessary for exporting NPM mutants; thedestruction of NES structure in NPM mutant A (NPM mut A no-NES) preventsexportation from nucleus so that the mutant appears localized in thenucleoplasm both in presence and in absence of LMB; the NPM mutant Ewhich possesses tryptophan 288 but lacks 290 one, repositions partiallyin nucleoli after treatment with LMB; the reinsertion of tryptophan 290(NPMmutA, A290W) produced about the same effect; when both tryptophans(288 and 290) have been inserted again in mutant A (C288W+A290W) thecorresponding protein fused to eGFP, despite the presence of NES, isexclusively localized in nucleoli, both in presence and in absence ofLMB;

FIG. 11A shows co-transfection experiments of murine cells with mutant AGFP-NPM and wild-type HA-NPM; NPM mutant acts as negative dominantversus wild-type NPM determining its partial moving in cytoplasm;

FIG. 11B shows Western blot with α-NPM or α-HA (left panel) andimmunoprecipitation with an anti-Flag antibody and Western blot withα-NPM or α-HA (right panel); the biochemical data confirm those oflabelling with fluorescent proteins;

FIG. 11C shows a hypothetical scheme of the mechanism ofnucleus-cytoplasma altered localization of NPM mutant A and wild-typeNPM, white not colored dots: tryptophan residues; dark dots: mutatedtryptophans; square: Nuclear Export Signal (NES);

FIG. 12 shows the scheme of a NPM immunostaining approach for studyingthe acute myeloid leukaemia in which the cases are respectively dividedin two groups for cytoplasmic (NPMc+) or nuclear (NPMc−) NPM expression;

FIG. 13 illustrates probe and primers used in RQ-PCR; two specificsystems for mutations A and B are shown; the amplification strategy usesa primer forward (cNPM1-F) and a probe (c.Probe) common to both systems;the primer forward is positioned in exon 11 while the probe in thejunction between exon 11 and exon 12; two reverses are cNPM-mut.A-R andcNPM-mut.B-R mutation-specific and are both designed in exon 12; thetable indicates primers and probe sequences;

FIG. 14 shows the amplification plot in Plasmidic RQ-PCR related to 5plasmidic dilutions tested in duplicate; the plasmid contains thesequence of NPM1 gene with the mutation A; the table shows the datareproducibility in further 4 experiments;

FIG. 15 shows the amplification plot of cDNA in RQ-PCR related to sixserial dilutions on the strength of a factor equal to 10 of a sample ofNPMc+ LAM with mutation A at diagnosis; the standard curve underlines“inclination”, correlation coefficient and intercept with Y-axis; thetable shows the diagnosis results achieved in 4 samples with mutation Aand in 1 with mutation B; in all samples a “Maximum reproduciblesensitivity” of 10⁻⁴ has been obtained;

FIG. 16 shows the monitoring of the mutated copy number of NPM, atdiagnosis and after the induction treatment in 13 patients with NPMc+LAM with mutation A (copy number expressed as copy number of NPM withmutation A/10000 copies of ABL);

(Abbreviation: CR=complete remission, PR=partial remission, NR=NotResponse);

FIG. 17 shows the monitoring of mutated copy number of NPM, at diagnosisafter the induction treatment and during the follow-up in 3 patientswith NPMc+ LAM mutation A; the RQ-PCR shows a different kinetic in thediminution of copy number; within 50 day therapy all patients evidencedcomplete hematological remission but exhibited a different mutated copynumber of NPM1; the patient I (square) shows a persistent hematologicalremission; a minimum mutated copy number is reached 90 days after thetherapy and remains the same during the follow-up; in the patient 11(diamond) the mutated copy number of NPM1 reaches a minimum around 100days after the therapy; a decisive increase of the copy number isevident during the follow-up along with the hematological relapse around200 days after the therapy; in the patient III (triangle) the mutatedcopy number of NPM1 reaches a minimum around 140 days after the therapy;a decisive increase of the copy number is evident during the follow-upalong with the hematological relapse (not represented in figure) thatoccurred about one month apart (there isn't the molecular datum ofrelapse); (abbreviation: CR=complete remission).

EXAMPLE 1 Study of Gene Expression of NPM and of Diagnostic andPrognostic Value Thereof in LAMs

A study that allowed to identify a large subgroup of acute myeloidleukaemias (about a third of the LAMs in the adult) characterized by thecytoplasmic NPM localization in leukaemic blasts, mutations of NPM gene,high frequency of normal karyotype, and good response to the inductionchemotherapy, has been carried out.

Materials and Methods Tumour Samples and Patients

The immunohistochemical studies have been carried out on 1845 paraffinembedded tumour samples. LAMs include: 591 primary LAMs (age 15-60,excluded M3 subgroup) of GIMEMA/EORTC study AML12 and 70 acutepromyelocytic leukaemias, 69 primary and 135 secondary LAMs outprotocol. The remaining samples represent haemopoietic andextra-haemopoietic tumours other than LAM.

With informed approval, the osteomedullary biopsy of each patient withLAM has been fixed in B5, transferred in 70% alcohol, decalcified andincluded in paraffin.

Antibodies

Monoclonal antibodies against NPM (Cordell et al., 1999; Falini et al.,2002), that allow the protein identification on routine paraffinembedded sections have been employed. The biopsy samples have been alsostained with monoclonal antibodies against the following molecules:nucleolin (C23) (Saint Cruz Biotechnology Inc., CA, USA), glicophorin,CD34 (DakoCytomation, Glostrup, Denmark), CD133 (Miltenyi Biotech,Bologna, Italy), and ALK (Falini et al., 1999).

Immunohistochemical Staining

Immunostaining has been carried out using the APAAP technique (Cordellet al., 1984). The sub-cellular NPM distribution (nucleus vs cytoplasm)has been assessed not knowing FAB subtype, cytogenetic or molecularbiology. The cases have been classified as NPMc+ (positive cytoplasm) orNPMc− (negative cytoplasm). All cases have been stained in parallel fornucleolin (C23) that, as expected, in case of NPMc+, has shown arestricted nuclear expression.

Molecular and Cytogenetic Analysis

The cytogenetic investigations have been carried out after short-termcultures. The karyotypes have been analyzed after G-banding and havebeen described according to Human Cytogenetic Nomenclature of theInternational System (Mitelman, 1995). The FISH investigations have beencarried out as previously described (Crescenzi et al., 2000).

The RT-PCR for the major fusion transcripts (PML-RARα, AML1-ETO,CBFB-MYH11, BC R-ABL and DEK-CAN), Southern Blotting, FISH analysis forMLL re-arrangements and analysis for FLT3 (ITD and D835) and MLL-ITDmutations have been carried out as previously described (Van Dongen etal., 1999; Soekarman et al., 1992; Noguera et al., 2002; tip et al.,1995).

Analysis of NPM Mutations

In the present study, the mutation analysis of NPM gene has been carriedout in 161 cases of lympho-haemopoietic malignant tumours: 52 NPMc+LAMs, NPMc-56 LAMs, 9 chronic myeloid leukaemias (CML), 7 acutelymphoblastic leukaemias (ALL) and 37 lymphoid neoplasias. Five patientswith NPMc+ LAM have been analyzed both at diagnosis and in completeremission step after chemotherapy.

For analysis of the NPM coding region, one microgram RNA wasretrotranscribed using the RT-PCR Thermoscript System (InvitrogenCorporation, Carlsbad, Calif., USA) and cDNA sequences were amplifiedwith primers NPM1_(—)25F, 5′-GGT TGT TCT CTG GAG GAG CGT TC-3′ andNPM1_(—)1112R, 5′-CCT GGA CAA CAT TTA TCA AAC ACG GTA-3′ using ExpandHigh-Fidelity Plus PCR system (Roche Applied Science, Mannheim,Germany).

In order to amplify exon 12 sequences from genomic DNA, twooligonucleotides were designed which specifically anneal to the flankingintron sequence regions (NPM1-F: 5′-TTA ACT CTC TGG TGG TAG AAT GM-3′and NPM1-R: 5′-CM GAG TAT TTG CCA TTC CTA AC-3′). PCR products werepurified according to standard methods and sequenced directly from bothends. All mutations were confirmed in independent PCR products and, inrepresentative cases, by cloning in pGEM-T Easy (Promega, Madison, Wis.,USA) and sequencing.

Relationships Between NPM Mutations and Other Mutations

The normal karyotype LAM cases with NPM mutations of AMLCG 99 protocolwere analyzed for the presence of other mutations, particularlyFLT3-ITD, FLT3-D835, MLL-PTD, CEBPA, NRAS, and KIT.

Expression Vectors: Plasmid-Construction

To follow sub-cellular destiny of mutated and wild-type NPM protein intransfection experiments, plasmids expressing wild-type (pEGFPd-NPMwt)or mutant (pEGFPd-NPMmA) NPM alleles fused to the Enhanced GreenFluorescent Protein (EGFP) were generated. To this end, NPM cDNAsequences were amplified from a NPMc+ LAM patient (code 497A/30)carrying a heterozygous mutation in the gene exon 12 with primersNPM189F_BamHI, 5′GCC ACG GAT CCG AAG ATT CGA TGG AC3′, andNPM1_(—)1044R_EcoRI, 5′ATC AAG AAT TCC AGA AAT GAA ATA AGA CG3′, andcloned in frame into pEGFP-C1 vector (BD Biosciences Clontech, PaloAlto, Calif., USA). Sequencing analysis confirmed the absence ofTaq-introduced errors in both plasmids.

Transient Transfection Experiments

NIH 3T3 cells were transiently transfected with pEGFPd-NPMwt,pEGFPd-NPMmA and empty pEGFP-C1 vector using the Lipofectamin 2000reagents (Invitrogen). Transfection efficiency was monitored by Westernblotting. Images were obtained with a BioRad MRC 1024 confocalmicroscope after nuclei counter-staining with propidium iodide. Theconfocal slices were obtained with a SCI Octane workstation and thereconstruction of the images starting from confocal slices was beencarried out using “Shadow Projection” module of Imaris software(Bitplane, Zurich, CH).

Statistic Analysis

Chi-square analysis in two-way contingency tables discloses theassociation between categorical variables. Statistical differencesbetween means were analyzed by t-test. The relationship between NPMlocalization and FLT3-ITD/D835 mutations, adjusted for age andcytogenetics, was investigated applying a logistic regression model withthese factors as independent variables and NPM as dependent variable.Cases with major translocations were excluded because their absoluteassociation with nucleus-restricted NPM expression don't provide a validparameter estimate.

The association between clinical and biological features (white bloodcell count at presentation, NPM sub-cellular expression, FLT3 mutations)and response to induction therapy was valued in 126 normal karyotype LAM(79 NPMc+ and 47 NPMc−) treated according to the GIMEMA LAM99P protocol.A multivariate Logistic model was applied. In all the statistic analysesin two-way, two values of p<0.05 were considered of statisticimportance.

Results Cytoplasmic Localization of NPM

Cytoplasmic NPM expression, defined as NPMc+ (FIG. 1A), was found in208/591 (35.2%) primary LAM cases of the GIMEMA/EORTC study (FIG. 1B).Such find appears specific for primary LAMs, not being detectable insecondary LAMs and in other human tumours that show always an exclusivenuclear NPM expression (FIG. 1B). In NPMc+ leukaemic cells, nucleolin(C23), another nucleolar antigen, retains its nucleus-restrictedlocalization as showed in FIG. 1A. In NPMc+ LAMs, the anomalous NPMexpression is usually found in almost all leukaemic cells; except incases of M5b (monocytic leukaemia), where cytoplasmic NPM is expressedonly in a variable percent of the neoplastic population, namely 30-60%of leukaemic cells, preferentially those more immature (monoblasts). Incontrast, NPMc− LAMs rarely contain NPMc+ leukaemic cells, mostlymitotic elements as shown in FIG. 1A on the right.

The anomalous cytoplasmic NPM expression of leukaemic cells must beconsidered a long-term event, as documented by its comparison in 25patients with NPMc+ LAM in relapse of disease up to 3 years after theinitial diagnosis.

Morphology of NPMc+ LAM

Morphology of NPMc+ LAM

The anomalous cytoplasmic NPM expression was found in all FABcategories, except M3 (acute promyelocytic leukaemia), M4eo (acutemyelomonocytic leukaemia with eosinophilia), and M7 (acutemegakaryocytic leukaemia) as shown in FIG. 1C. Particularly, the figureshows the correlation between NPM sub-cellular expression and morphologyin 591 primary LAMs of GIMEMA/EORTC study plus 70 acute promyelocyticleukaemias with t(15;17) (out of protocol). The frequency of cytoplasmicNPM expression ranged from 13.6% in M0 (minimally differentiated LAM) to93.7% in M5b (acute monocytic leukaemia). Most NPMc+ LAM of M5b and M6(acute erythroid leukaemia) type and about 30% of NPMc+ M1 (LAM withoutmaturation), M2 (LAM with maturation) and M4 cases are characterized forthe presence of cytoplasmic NPM not only in myeloid blasts but also inerythroid precursors, particularly in the proerythroblasts (FIG. 1D)and, less frequently, in the megakaryocytes (not shown data).

The presence of cytoplasmic NPM in different hematopoietic lineages ledto the expression analysis of some molecules, as CD34 and CD133antigens, which occur on hematopoietic stem cells. CD34-positivity(defined as 20% positive cells) was detected in 12/159 (7.5%) NMPc+ LAMand in 227/317 (71.6%) NMPc− LAM (p<0.001) as shown in FIGS. 2A-2E.Therefore NPMc+ appears mutually exclusive with CD34 and CD133expression. CD34-negative NMPc+ LAMs characteristically also lackingCD133, as shown in FIG. 2F.

The wide morphological spectrum and the involvement of varioushematopoietic lineages of the NPMc+ LAM reflects a possible originthereof from CD34−/CD38− rare hematopoietic stem cells, which wereidentified in several animal species, included murine and human species(Goodell et al., 1997; Engelhardt et al., 2002). Alternatively, CD34could be unregulated as effect of the leukaemic transformation.

Karyotype of NMPc+ LAM

Cytogenetic data are available in 493/591 cases of LAM (166 NPMc+ and327 NPMc−). Over 85% ( 142/166) of NPMc+ LAM has a normal karyotype asshown in FIG. 3A. In contrast, only 26.9% ( 88/327 cases) of NPMc− LAMhas a normal karyotype (p<0.001). Overall, 61.7% of normal karyotype LAMare NPMc+ ( 142/230 cases) as shown in FIG. 3B. 24 NPMc+ LAM cases showthe presence of smaller chromosomal translocations and, among these, 12(50%) cases have both normal and abnormal metaphases.

None of the LAM cases carrying major genetic abnormalities is NPMc+, asshown in FIGS. 3B, 3C and 3D.

FLT3-ITD and Other Genes Mutations in NPMc+ LAM

FLT3, ITD or D835 mutations were detected in 59/219 (26.9%) and 13/202(6.4%) LAM cases with a normal karyotype, respectively; one case iscarrier of both ITD and D835 mutations. FLT3-ITD mutation was 2.5 foldmore frequent in NPMc+ cases than NPMc− cases (p<0.003), as shown inhistograms of FIG. 3E. A multivariate logistic regression model adjustedfor age and cytogenetics allows to establish an independent associationbetween cytoplasmic NPM (dependent variable) and FLT3-ITD, as shown inFIG. 3G. No statistical association is observed between FLT3-D835mutations and NPM sub-cellular localization (p=0.5), possibly because oflow number of D835-mutation cases.

In a subsequent study on cases of AMLCG 99 protocol (Schnittger et al.,2005), an increased FLT3-ITD mutation frequency in LAM cases with NPMmutation was confirmed. In contrast, NPM mutation is rarely associatedto MLL (MLL-PTD) and CEPBA mutations.

Response to Induction Therapy of NPMc+ LAM

Response to induction therapy was evaluated in 126 LAM cases with normalkaryotype (79 NPMc+ and 47 NPMc−) treated according to the GIMEMA LAM99Pprotocol. In table 1 is shown logistic regression model of the responseto induction therapy in 126 patients.

TABLE 1 Analysis of maximum-likelihood assessment Odds Ratio Assess Wald95 OR D Stand. Chi- Pr > Chi- Confidence Parameter F Assess Error squaresquare OR range Intercept 1 0.9596 0.3761 6.5112 0.0107 FLT3 1 −0.40200.4541 0.7836 0.3760 0.669 0.2 1.6 (M vs U)* NPM 1 1.0946 0.4647 5.54860.0185 2.988 1.2 7.4 (Cit vs Nucl)^(#) WBC 1 −1.3123 0.4748 7.63990.0057 0.269 0.1 0.6 (≦80 vs >80) Age 1 −0.3405 0.4498 0.5732 0.44900.711 0.2 1.7 (≦48 vs >48) *M = Mutated; U = Not mutated; WBC: number ofwhite blood cells; ^(#)Cit. = cytoplasmic positivity; Nucl. =nucleus-restricted positivity.

The multivariate model for complete response achievement includes whiteblood cell count (categorized at the 75^(th) percentile), age(categorized at median value), NPM sub-cellular expression and FLT-3mutations. Analysis is shown in table 1 and shows that the white bloodcell count and NPM are both independent prognostic factors. Particularlyas negative prognostic factor impinges a white blood cell count over80×10⁹/L (p<0.006; OR=0.269; CI 95%: 0.1-0.6) and as positive prognosticfactor the fact that leukaemic cells have cytoplasmic NPM (p<0.019;OR=2.988; CI 95%: 1.2-7.4).

NPM Mutations as Prognostic Factor in Normal Karyotype LAMs

According to the presence of NPM gene and FLT3-ITD gene mutations,normal karyotype LAMs can be divided in 4 subgroups: mutated NPM/mutatedFLT3, mutated NPM/not mutated FLT3, not mutated NPM/mutated FLT3 and notmutated NPM/not mutated FLT3. The analysis carried out by the authors on401 LAM cases with normal karyotype (Schnittger et al, 2005) shows thatthe presence of a NPM mutation in absence of a FLT3 mutation identifiesa subgroup of leukaemias with more favorable prognosis. Similar resultswere reported subsequently also in other two studies (Dhoner et al.,2005; Verhaak et al., 2005).

Analysis of NPM Mutations in LAMs and in Other Tumours

NPMc+ LAM doesn't express NPM-ALK, NPM-RARα, NPM-MLF1 fusion proteins,or other fusion proteins containing NPM, as shown by absence ofrespective fusion genes upon FISH, absence of respective fusiontranscripts upon RT-PCR, absence of said fusion proteins uponimmunohistochemistry, exclusive presence of 38 kDa NPM polypeptide uponWestern Blotting, and demonstration of cytoplasmic NPM through fourdifferent anti-NPM monoclonal antibodies.

In the present study, analysis of the NPM coding region, carried out byRT-PCR and direct sequencing, revealed mutations affecting exon 12 inall but one NPMc+ case. FIG. 4A is a schematic representation of the NPMgene, and mutations are summarized in FIG. 4B. In total, six differentsequence variants were observed, all of them leading to a frameshift inthe region encoding for the carboxyl-terminus domain of the NPM protein.The most frequent mutation (type A: gatctctgTCTGgcagtggaggaagtctctttaagaaaatag) is a duplication of a TCTG tetranucleotide atpositions 956-959 of the reference oligonucleotide sequence NM_(—)002520(GenBank) as shown in FIG. 4C; the resulting shift in the reading frameis predicted to alter the C-terminal portion of the NPM protein bysubstituting the last seven amino acids (WQWRKSL) with 11 differentresidues (CLAVEEVSLRK). Three additional mutations (type B:gatctctgCATGgcagtggaggaagtct cttaagaaaatag, type C:gatctctgCGTGgcagtggaggaagtct ctttaagaaaatag, and type D:gatctctgCCTGgcagtggaggaagtct ctttaagaaaatag) include distinct 4-basepair insertion at position 960, all resulting in the same frameshift asmutation A. In the last two mutations (type E: gatctctggcagtCTCTTGCCCaagtctctttaagaaaatag and type F: gatctctggcagtCCCTGGAGAaagtctctttaagaaaatag), nucleotides 965-969 (GGAGG) are deleted andin place of them, two different 9-base pair sequences are inserted, notmodifying the frameshift and creating a new carboxyl-terminus domain of9 amino acids. Regardless of the specific type of mutation, the mutantsare characterized by replacement of at least one of the two tryptophanresidues (W) that in wild-type sequence are at position 288 and 290. Theobtained results are according to previous evidences in studies carriedout on mice that show the importance of tryptophan amino acid fornucleolar localization (Nishimura et al., 2002). In addition, all mutantproteins shared the same last 5 amino acid residues VSLRK. Thus, despitegenetic heterogeneity, all NPM gene mutations result in a novel sequencein place of the NPM protein C-terminus.

The presence of mutations in the NPM exon 12 and their specificassociation with NPMc+ LAM was confirmed in 11 samples also bysequencing analysis of genomic DNA. NPM mutations are heterozygous andoccur only in the malignant clone, as they are not present in bonemarrow samples from patients in complete remission (N=5) afterchemotherapy. Mutations are observed in NPMc+ LAM of all FAB categories,included NPMc+ cases with abnormal karyotype or CD34 expression.Conversely, all 56 of the NPMc-LAMs as well as 53 malignancies otherthan LAM, and NPMc− display wild-type NPM sequences as shown in table 2.

TABLE 2 Mut. Mut. Tumour type N. FAB CD34+ FLT3 NPM LAM NPMc+ 52 normalkaryotype 49 All*  2/49 25/46 48/49 Abnormal karyotype  3 M1, M5b 1/31/3 3/3 LAM NPMc− 56 normal karyotype 12 M1-M6  8/10  4/12  0/12Abnormal karyotype** 44 M1-M6 5/8 3/8   0/44*** LMC  9 n.a. 0/9 n.d. 0/9Lymphoid neoplasias  44^(#) n.a. n.d. n.d.  0/44 *Except for M3, M4eoand M7. **includes 7 t(15; 17 s); 12 t(8; 21 s); 13 Inv(16); 1 MLLrearrangement; 1 inv(3; 1 t(6; 9), a complex karyotype; ***Case with Inv16 that shows a deletion of three nucleotides in exon 6 of NPM1 atpositions 583-585 without involving 3′ terminal. ^(#)includes: 7 acutelymphoblastic leukaemias; 7 lymphocyte B chronic lymphatic leukaemias; 5mantellari lymphomas; 5 follicular lymphomas; 10 big cells B lymphomas;5 Burkitt lymphomas; 5 multiple myelomas. Mut.: mutated; n.a.: notapplicable; n.d.: undone.

In subsequent studies, the authors confirmed afore described results for1009 LAM cases subjected to sequencing of NPM gene. FIG. 6 shows 40 NPMmutations till now individualized.

Transfection of the Mutated NPM Gene

To confirm whether the NPM exon 12 mutations are responsive forcytoplasmic dislocation of the NPM protein, NIH 3T3 cells weretransiently transfected with expression vectors encoding for thewild-type and mutant allele fused with EGFP. Confocal microscopy showedthe expected nucleolar localization for the EGFP-NPM wild-type protein,whereas the NPM mutant isoform is clearly dislocated into the cytoplasmas shown in FIG. 4D.

These results indicate clearly a causal correlation between the geneticevent (NPM mutations) and the cytoplasmic localization of the NPMprotein. In addiction, the fact that mutated NPM is closely associatedwith the normal karyotype and is not seen in leukaemias with majorcytogenetic abnormalities suggests that the NPM mutation is the primaryleukaemogenic event. The mutation might interfere with the normalfunction of NPM, such as, for example, interaction with the Arf or p53protein, (Colombo et al., 2002; Kurki et al., 2004; Horn et al., 2004).Mutations might also perturb other NPM functions that have been mappedwithin C-terminal domain, such as nucleic acid binding (Hingorani etal., 2000), ATP (Chang et al., 1998), DNA-polymerase alpha-stimulatoryactivity, or binding with the tumour suppressor gene Arf (Bertwistle etal, 2004).

EXAMPLE 2 Production of Specific Antibodies Against PolypeptideSequences of C-Terminal of the Leukaemia-Specific NPM

The polypeptide sequences represent ideal immunogenes for the productionof specific antibodies that include all types of polyclonal andmonoclonal antibodies, human monoclonal antibodies, and humanizedantibodies produced by genetic recombination techniques.

The peptides corresponding to A, B, C, D, E and F sequences of FIG. 4Bcan be synthesized chemically according to standard procedures.

Every animal species is employable for antibody preparation. Methods forthe antibody production and immunization procedure of animal species(inoculation routes of antigen, use of Freund adjuvant to increase theimmunogenetics of injected mixture, frequency of immunizations, etc) arelargely described in the scientific literature.

Monoclonals

Balb/c Mice can be inoculated by intraperitoneal route with specificpeptides bound to KLH (3 immunizations of 150 micro grams of peptideevery 10 days). Such immunization program is followed by an intravenousbooster (150 micro grams), three days before the fusion, with thepurpose to increase immune response to the maximum.

Monoclonal antibodies can be produced with the “hybridoma method”(Goding J., 1983) that consists in the formation of hybrid cellsresulting from spleen normal cells with myeloma cells. The authors ofthe present invention use the P3-NS1-Ag-4-1 myeloma lineage (abbreviatedas NS-1) provided them from Prof. David Y Mason laboratory, Oxford, andderiving from P3K lineage, which synthesizes only a light chains thatare not secreted but degraded internally and lack HGPRT enzyme.

Following the cellular fusion, the hybrid selection is carried out withaddition of hypoxanthine, aminopterin and thymidine (HAT) to the medium.The only cells able to survive under this condition possess:

1. myeloma neoplastic characteristic of growing in vitro;2. hypoxanthine guanidine phosphoribosil transferase enzyme (HGPRT) ofspleen cells that allows them to use hypoxanthine and thymidine forsynthesizing nucleotides and therefore DNA.

Not fused myeloma cells die because the lack HGPRT enzyme and thereforecannot use hypoxanthine for biosynthesizing nucleotides, while spleencells die (even if they are HGPRT+) because they are unable to grow invitro.

For purposes of the present research, the hybridoma supernatant can betested directly on cytological samples or on paraffin embedded sectionsof LAM samples containing mutated form of NPM (NPMc+ LAM) and normalform (wild-type) of NPM (NPMc− LAM). The rational criterion of screeningis the identification of hybridomas producing specific monoclonalantibodies against peptides of the invention (corresponding toC-terminal of NPM), namely antibodies reacting with NPMc+ LAM but notwith NPMc−. Such antibodies have the advantage that can be used both oncytological samples (smears and cytocentrifugates) and on paraffinembedded tissue sections and, in addiction, both for diagnostic purposeand for monitoring leukaemic minimal residual disease. Afteridentification, hybrids can be cloned according to well known methods(“limiting dilution”) and grown in vitro in large amounts. Clones can bethen cryostored in liquid nitrogen so that to have an available “bank”of the aforesaid antibodies.

Polyclonal Antibodies

Polyclonal antibodies against peptides of the present invention(specific sequences of C-terminal of mutated NPM) can be produced inseveral animal species. Polyclonal antibodies are referred both to wholeanimal serum containing such antibodies and to serum fractions enrichedin antibody.

Particularly, IgG or IgM serum fractions that contain only the specificantibodies for peptides of the present invention, can be obtained byeluting serum through a column containing bonded peptides of the presentinvention (affinity chromatography) and subsequently purifying thisfraction by a column containing protein A or protein G. These antibodieshave the advantage that can also be used on cytological samples (smearsand cytocentrifugates) and not only on paraffin embedded tissuesections, and for monitoring leukaemic minimal residual disease inaddiction to diagnostic purpose.

Production of Sil-C Polyclonal Antibody Materials and Methods

The Sil-C antibody was produced by immunizing the rabbits with a 11amino acid synthetic peptide (NHCOCH3-CLAVEEVSLRK-COOH) (Inbio Ltd,Tallin, Estonia) which comprises two mutated tryptophans, a NES portionand the VSLRK peptide of mutant A. The rabbit sera were purified byaffinity chromatography using columns containing the peptide used asimmunogen (elution with 0.23 M Tris, 0.2 M Na₂HPO₄, pH 0.8). Thereactivity against the peptide was shown by ELISA technique.Particularly, the ELISA plates were adsorbed overnight at 4° C. with 50μl of peptide at a concentration of 10 μg/ml in TBS. After the blockingwith PBS containing 3% bovine fetal serum, the rabbit serums were addedat increasing dilutions to the wells (from 1/100 up to 1/72900). Afterthe washing in PBS-Tween 20 (0.05%), to each well were added 50 μl of asecondary goat antibody conjugated with peroxidase (dilution 1/5000 inPBS).

The antibody-antigen reaction was observed adding to each well 50 μl ofO-phenylenediamine dihydrochloride. The reading was carried out at 492nm. Pre-immune serums were used as negative control. The biochemicalcharacterization of antibodies was carried out with standard Westernblotting, immunoprecipitation and co-precipitation techniques.

Results

A polyclonal antibody (named Sil-C), which recognizes specifically anantigenic epitope comprising two mutated tryptophans, a NES portion andthe VSLRK peptide belonging to mutated nucleophosmin proteins (NPM) asdefined in attached claims 1-6, was produced.

In Western blotting experiments, Sil-C antibody recognizes a 37 kDAspecific band only in whole lysed cells from leukaemias with NPMmutations (NPMc+ LAM) (patients 1-3) but not from patients with LAMwithout NPM mutations (NPMc−) (cases 4-6) (FIG. 5A, on top in the left).In contrast, the monoclonal anti-NPM 376 antibody reacting with bothwild-type and mutated NPM, identifies a 37 kDA band in all testedleukaemic patients, regardless of the presence or not of mutations ofNPM gene (FIG. 5A, in bottom on the left). In patient No. 2, carrier ofa NPM mutation type A, Sil-C antibody recognizes a 37 kDa band only inthe cytoplasmic fraction of leukaemic cells (FIG. 5B, on top in theright). The picture, as expected, differs from that of the monoclonal376 antibody which identifies a band with the same molecular weight,both in lysed cytoplasmic fraction and in those of the nuclear fraction(FIG. 5B, in bottom in the right).

These biochemical data were also confirmed at cytological level byimmunohistochemical staining of medullary biopsies from 10 patients withNPMc+ LAM. In these samples, the monoclonal anti-NPM antibodies,recognizing both normal and mutated NPM, identify NPM both in nucleus,and in cytoplasm (FIG. 5E). In contrast, Sil-C antibody identifiesexclusively NPM protein at cytoplasmic level (FIG. 5D). The specificityof this reaction is clearly shown by the fact that: i) it is completelyabolished from the pre-incubation of Sil-C antibody with the peptideused as immunogen; ii) Sil-C antibody doesn't stain biopsy samples ofpatients with NPMc− LAM, namely lacking NPM mutations (FIGS. 5F and 5G).

These results show that Sil-C antibody recognizes specifically NPMmutants and that the mutants are restricted exclusively in the cytoplasmof leukaemic cells that containing NPM mutations. The availability ofspecific antibodies against leukaemic NPM mutants opens new perspectivesfrom a diagnostic and therapeutic standpoint.

From a diagnostic standpoint, these reagents could be employed for thediagnosis at the beginning and also for monitoring minimal residualdisease in combination with techniques of quantitative PCR (see below).

From a therapeutic standpoint, it is supposable the use of intracellularantibodies (“intrabodies”) able to inhibit the leukaemic NPM mutatedproteins without damaging the function of physiological NPM protein.

EXAMPLE 3 Preparation of Vaccines

The vaccines can be administered in formulations recognized by “T cellreceptor” (mononuclear cells from peripheral blood) or presented byanti-gen-presenting cells (e.g. dendritic cells, cells B, macrophages).

In this context the term “vaccine” means any substance or compound thatserves to induce anti-tumour immunity anti-tumour immunity meanscytotoxic responses (T cellular), induction of antibodies thatrecognizes tumour cells and production of cytokines with anti-neoplasticactivity. The anti-tumour activity can be measured in vitro(cytotoxicity) or in vivo in experimental animal models.

Efficacy of the anti-tumour vaccines is known to be increased whenvarious polypeptides, in combination, having different structures, areused. Therefore to that end anti-LAM NPMc+ vaccines can containdifferent synthesis polypeptides with different specificity andsequences provided they induce the recognition of tumour cellscontaining NPM gene mutations.

The vaccine of this invention can be conjugated with immunogenicmolecules universally known as carriers.

It can contain, in its formulation, suitable solutions for inoculum(physiological saline, various saline solutions) and excipients.

In addition, the vaccine can contain or be administered with adjuvants,namely any molecule with immunostimulant activity. The adjuvantadministration can be carried out in any time point preceding orfollowing inoculum of anti-tumour vaccine.

The vaccine of the present invention can be administered by systemic orlocal route in single or multiple dose.

The evaluation of the immune response will be carried out according tothe methods well known and described in scientific literature.Particularly, after vaccine inoculum, anti-gen epitopes are presented toB and T cells by anti-gen-presenting cells.

Therefore the determination of cytotoxic responses can be carried out onboth CD4+ and CD8+ cells T, and all cellular populations able to inducecytolysis or apoptosis (e.g. neutrophiles, NK cells). Specifically, theactivation, (immunophenotype), proliferation ability, (by methods ofincorporation of radioactive markers), cytotoxic ability on opportunelyprepared targets, ability to secrete cytokines (ELISA, ELISPOT methods)thereof can be measured.

As to antibody responses these can be measured in vitro or in vivo withexperiments of serum transfer and inhibition of the tumour growth.

The efficacy in vivo in animal models can be measured according tostatistic significance criterions verifying the anti-tumour response inopportunely designed control groups.

Object of the evaluation will be survival, measurement of tumourbiomarkers, inhibition of the growth of tumour cells, regression oftumour masses, reduction of tumour-induction ability.

EXAMPLE 4 Study of Mechanism of Cytoplasmic Accumulation of NPM Mutants

The present applicants elucidated the molecular mechanism that leads tothe aberrant cytoplasmic accumulation of NPM in NPMc+ acute myeloidleukaemia.

Materials and Methods

Cells for transfection, AML samples and OCI/AML3 cellular line Fortransfection experiments, NIH-3T3 and H1299 cells were used. Leukaemiccells from 5 leukaemic patients (3 of which carrying the mutation A ofNPM and 2 carrying wild-type NPM gene), were isolated by separation withFicoll-Hypaque and used for biochemical studies and confocal microscopeanalysis. Biopsies of bone marrow (n=373) and pellet of blasts ofperipheral blood (n=20), from 393 patients with AML of the GIMEMA AML99P and GIMEMA AML12/EORTC protocol, were fixed in B5 and included inparaffin. OCI/AML3 cellular line, which we identified as the onlycontaining the mutation A in exon 12 of NPM gene (among 79 testedmyeloid human lines) (Quentmeier et al., 2005) was grown in alpha-MEMwith 10% FBS plus glutamine and antibiotics at standard concentrations.

Mutational Analysis of NPM Gene

Studies were carried out on leukaemic cells of 393 adult ALM patients ofthe GIMEMA AML99P and GIMEMA AML12/EORTC protocol. The selection ofcases for mutational analysis was carried out based on materialavailability for NPM immunohistochemical identification. The mutationsof exon 12 of NPM gene were analysed with RT-PCR and sequencing aspreviously described or using DHPLC (Wave™ System, Transgenomic Inc.,Omaha, Nebr.; USA).

Plasmid Construction

The mutants A, B, C, and D of NPM were produced by PCR using NPMwt astemplate; the same forward primer (5′ CGC CAC GCT AGC GAA GAT TCG ATGGAC) was used and a different reverse primer for each mutant (mutantA-5′: CTA TTT TCT TAA AGA GAC TTC CTC CAC TGC CAG ACA GAG ATC TTG AATAGC CTC TTG G; mutant B-5′: CTA TTT TCT TAA AGA GAC TTC CTC CAC TGC CATGCA GAG ATC TTG AAT AGC CTC TTG G; mutant C-5′: CTA TTT TCT TAA AGA GACTTC CTC CAC TGC CAC GCA GAG ATC TTG AAT AGC CTC TTG G; mutant D-5′: CTATTT TCT TAA AGA GAC TTC CTC CAC TGC CAG GCA GAG ATC TTG AAT AGC CTC TTGG). The products of the respective PCRs were cloned inpcDNA3.1/NT-GFP-TOPO (Invitrogen, Carlsbad; Calif., USA), and checkedwith insert sequencing. To produce the double N-terminal flag-HA tag inplasmids with wt NPM and mutation A, a PCR was carried out using astemplate wild-type NPM or mutant A; as forward and reverse primer wererespectively used 5′ CGC CAC GCT AGC GAA GAT TCG ATG GAC and 5′ TCA AGAATT CCA GAA ATG AAA TAA GAC. The PCR product was digested using NheI andECORI, and the fragment was sub-cloned in PCIN4 vector, containing theFlag-HA tag at N-terminal end of the fragment. The precision ofFlag-HA-NPM-wt and Flag-HA-NPM-mutant A sequences was confirmed bysequencing.

NPM mutants E, G and R were produced through QuikChange FineSite-Directed Mutagenesis Kit (Stratagene, You Jolla, Calif.), using astemplate pEGFP-C1-A/PMwt, and according to manufacturer instructions.Primers were designed on the followings sequences:

NPM_MUT _E: 5′-GATCTCTGGCAGTCTCTTGCCCAAGTCTCTTTAAG-3′; NPM_MUT_G:5′-GATCTCTGGCAGTGCTTCGCCCAAGTCTCTTTAAG-3′; NPM_MUT_R:5′-GATCTCTGGCAGAGGATGGAGGAAGTCTCTTTAAG-3′.

NPM_MUT_A A290W, NPM_MUT_A C288W+A290W, e NPM_MUT_A_NO_NES plasmids wereproduced using pEGFP-C1-NPMmA as template, exploiting the localizationof mutagenesis sites between cutting sites of BglII and EcoRI enzymes.By using a partially complementary primer couple, containing the desiredmutation and protruding ends compatible with ends produced by thedigestion with BglII-EcoR, it was possible to ligate the double strandDNA produced by annealing primers to pEGFP-C1-NPMmA vector previouslydigested using the two above restriction enzymes.

The sequences of the used primers are:

NPM_MUT_A A290W_FOR: 5′-GATCTCTGTCTGTGGGTGGAGGAAGTCTC TTTAAGAAAATAGG-3′; NPM_MUT_A A290W_REV:5′-AATTCCTATTTTCTTAAAGAGACTTCCTCCACCCACAGACAGA-3′; NPM_MUT_A C288W+ A290W_FOR: 5′-GATCTCTGGCTGTGGGTGGAGG AAGTCTCTTTAAGAAAATAG G-3′;NPM_MUT_A C288W + A290W_REV:5′-AATTCCTATTTTCTTAAAGAGACTTCCTCCACCCACAGCCAGA-3′ NPM_MUT_A_NO_NES_FOR:5′-GATCTCTGTGGAGCAGGGGAGGAAG GCTCTTTAAGAAAATAG G-3′;NPM_MUT_A_NO_NES_REV: 5′-AATTCCTATTTTCTTAAAGAGCCTTCCTCCCCTGCTCCACAGA-3′.

Every construct was verified by sequencing.

Inhibition of the CRM1-Dependent Nuclear Exportation

The H1299 cells were seeded on the surface of six-well plates 24 hoursbefore transfection. 5 μg of expression vector encoding for wild typeHA-NPM, GFP-NPM-mutant A, or both, were transfected using theprecipitation method with calcium-phosphate. After 24 hours, the cellswere treated with 20 nM leptomycin B, a Crm1 specific inhibitor (Sigma,St. Louis, Mo., USA) for 8 hours or not treated, respectively. The cellsthen were fixed in 4% paraformaldehyde for the immunofluorescence study.

NIH-3T3 cells were transfected using Lipofectamin 2000 (InvitrogenCarlsbad, Calif., USA) following the manufacturer instructions. After 24hours, the cells grown on the slide were incubated with cycloeximide(Merck Biosciences Ltd, Nottingham UK) 10 micro grams/ml (30 minutes)and leptomycin B (Merck Biosciences Ltd, Nottingham UK) 20 ng/ml (5hours), or other Crm1 inhibitors such as ratjadons A and C (AlexisBiochemicals, Carlsbad, Calif., USA) 20 ng/ml (5 hours).

The cells were fixed in 4% paraformaldehyde (10 minutes) forimmunofluorescence and confocal microscope analysis.

For “time course” experiments, transfected cells were transferred insidean Attofluor chamber (Molecular Probes, Eugene, Oreg., USA) and wereobserved using a MRC-1024 confocal apparatus (Biorad Cambridge, UK)assembled on an Olympus IMT-2 microscope. The images of a single sectionwere recorded before and after the addition of leptomicin B, at 60second intervals, using the time-series function of Laser-Sharp program(BioRad). The excitation wavelength was 488 nm and images were detectedusing a filter from 505 to 550 nm on the PMT2.

The images were processed and analysed using the well known ImageJprogram (Rasband WS, Image J, U.S. National Institutes of Health,Bethesda, Md., USA, http://rsb.info.nih.gov/ij/, 1997-2005).

For western-blotting analyses of the sub-cellular distribution ofGFP-NPM mutant A protein, N1H-3T3 cells transfected with GFP vector orwith GFP-NPM mutant A, as described above, were incubated withleptomicin B 20 ng/ml (or with methanol as control) without cycloeximidefor 3 or 6 hours. Then cells were harvested, washed with PBS and lysedin hypotonic buffer according to the method of Schreiber et al. (1989).The supernatant was preserved as cytoplasmic fraction. The pellet,containing nuclei, was again washed with the hypotonic buffer, thensolubilised in a hypertonic buffer and boiled in a solution containingSDS before loading.

Equivalent dilutions (with the same cell number) of cytoplasmic andnuclear fractions were loaded, blotted on nitrocellulose and incubatedwith an anti-GFP monoclonal antibody (Roche, Indianapolis, Ind., USA)for Western-blot analysis of the distribution of GFP-NPM mutant A.

Cells deriving from two patients with NPMc+ AML (carrying the mutationA) and from OCI/AML3 cellular line were harvested in medium (10⁶ cell/mlin 24-well plates) and incubated at 37° C. with 5% CO₂ for 5 hours.After the incubation overnight with leptomycin B (20 ng/ml), the cellswere washed with PBS and centrifuged. The cell pellet was fixed in B5and paraffin for the immunostaining.

Immunostaining Procedures

The DAPI staining was used to visualise nucleuses of H1299 cellstransfected with the GFP-NPM-mutant A and with GFP-NPM wt. For Flag-HAconstructs, the fixed cells were made permeable with 0.2% Triton-X 100(10 minutes) followed by blocking with 10% anti-goat serum (30 minutes).Then primary anti-HA antibody was added (1:1000, Roche Indianapolis,Ind., USA) for 1 hour followed by incubation for 30 minutes with thesecondary Alexa-568 antibody (1:1000, Molecular Probes, Oregon, USA).The images were taken using a digital camera with the Spot 4.09 program(Diagnostic Instruments, Sterling Heights, Mich., USA).

The nuclei of NIH 3T3 cells transfected with GFP-NPMwt and GFP-NPMmutant A were stained with propidium iodide. The nucleolin staining(C23) of NIH-3T3 cells was carried out with the primary anti-nucleolinantibody purchased from DakoCytomation (Glostrup, Denmark), followed byTexas Red conjugated secondary antibody (Southern BiotechnologyAssociates, Birmingham, Ala.); the nuclei were contra-stained with theTO-PRO-3 (Molecular Probes, Oregon, USA).

The NPM coloration of paraffin embedded sections of AML cases containingthe mutation A was carried out with monoclonal anti-NPM antibodiesfollowed by a Alexa 488 conjugated secondary (Molecular Probes, Oregon,USA); the nuclei were contra-stained with propidium iodide. The imageswere taken with a confocal microscope Zeiss LSM 510 (Carl Zeiss, Jena,DE), using laser excitation wavelengths at 488 nm (for ALexa 488), 543nm (for Texas Red and the propidium iodide) and 633 nm (for TO-PRO-3),respectively. The laser intensity tuning, diameter of pinholes, andconfiguration of the light detection were set to achieve the bestsignal/noise ratio and avoid fluorescence crossover. The images werethen transferred to a SGI Octane workstation (Silicon graphics, MountainView, Calif., USA) for further elaboration; 3D reconstruction was madewith the shade or iso-surface method using the Imaris program (Bitplane,Zurich, CH).

NPM research on paraffin embedded sections using alkaline phosphatasemethod was done for 393 patients as previously described. The sampleswere classified as cytoplasmic or nuclear NPM without knowing theresults of mutational analysis.

Cellular Extracts, Western Blotting, and Co-Immunoprecipitation

The nuclear and cytoplasmic extracts were prepared according to themethod of Schreiber et al. (1989). For co-immunoprecipitationexperiments, the cells were lysed in 1 ml of ice-cold lysis buffer (1%NP-40, 150 mM NaCl, 25 mM Tris, pH 7.5, 1 mM EDTA, 1 mM Na₃VO₄ 1 μg/mlleupeptine, 1 μg/ml aprotinin and 1 mM PMSF). After 20 minutes of iceincubation, lysed products were centrifuged at 14,000×g for 10 minutes4° C. and incubated with 4 μg of an unspecific control IgG or specificpolyclonal rabbit anti-NPM, named Sil-C, or monoclonal mouse anti-NPM(Clone 376) antibody, respectively, and 30 ul of the Protein A/GPlus-agarose beads (Saint Cruz Biotechnology, Inc.) in incubationovernight at 4° C. The beads then were washed at least thrice with thewashing buffer (0.1% NP-40, 150 mM NaCl, 25 mM Tris pH 7.5, 1 mM EDTAand inhibitors). The proteins were separated on a SDS-polyacrylamide gel(SDS-PAGE) and transferred on a PVDF membrane (Millipore), where wereincubated with primary antibodies, namely rabbit polyclonal anti-Crm1(Saint Cruz Biotechnology, Inc.) or mouse monoclonal anti-Crm1 (BDTransduction Laboratories); respectively; after incubation with aHRP-conjugated secondary antibody, peptides recognized in Western blotwere detected using ECL method according to manufacturer instructions(Amersham Bioscience).

Results

The analysis of 40 mutations of NPM gene till now identified in thousandleukaemic patients (FIG. 6), shows that, despite their geneticheterogeneity, all mutations determine some common alterations at levelof the carboxyl-terminal portion of the corresponding mutated NPMproteins.

The FIG. 6 shows the changes in the tryptophans 288 and 290 and thecreation of a NES motif in 40 mutant NPM proteins identified inleukaemic patients; the mutation frequency (%) is present only for 393AML cases here studied for which were available, in addition to themolecular data, also the results of the immunohistochemical (1H)staining. Alterations are of two types: i) the mutation of both thetryptophans 288 and 290 (or only 290) and ii) the creation a new motifnamed NES (“Nuclear Export Signal motif). NES is a protein structurethat is specifically recognized by Crm1 (or Exportin 1), the proteinphysiologically delegated to the transport of other proteins fromnucleus to cytoplasm. From a molecular standpoint, NES motif is definedas a sequence of about 10 amino acids of type YxxxYxxYxY where Yindicates a hydrophobic amino acid of leucine, isoleucine, methionine,valine or phenylalanine type and x is equivalent to others amino acids.In the NES, the hydrophobic amino acids Y are spaced by preciseintervals (varying from 1 to three spaces), where the spacing isrepresented by other amino acids that, in the scheme, are indicated withthe x letter.

The NES type can vary from each other NPM mutant. The type and thefrequency of NES in the various leukaemic NPM mutants are illustrated inFIG. 6. The more frequent NES motif, found in about 65% of mutants, isdenominated LxxxVxxVxL (where L=leucine, V=valine and x is equivalent toother amino acids). The remaining 35% of NPM mutant proteins containsrarer NES variants, in which the valine in second position of NES isreplaced with another hydrophobic amino acid. Examples of this type,illustrated in FIG. 6, are NESs of type LxxxLxxVxL, LxxxFxxVxL,LxxxMxxVxL, LxxxCxxVxL (where L=Leucine; V=Valine; F=Phenylalanine;M=Methionine; C=Cysteine; and x is equivalent to another amino acid).

Relationship Between NES Type and Mutations of Tryptophans in Position288 and 290

From FIG. 6 can be deduced that tryptophan in position 290 is mutated inall 40 leukaemic NPM mutants. On the contrary, 13 of the 40 mutants(32.5%) preserve tryptophan in position 288. A careful analysis ofprotein structures of the 40 mutants clearly indicates that arelationship exists between NES type and mutations at level oftryptophans 288 and 290 (FIG. 6 and Table 3). Particularly it is shownas the more frequent NES motif, (namely LxxxVxxVxL type) is alwaysassociated to mutations of both tryptophans 288 and 290, whiletryptophan 288 is preserved only in NPM mutant proteins that contain aNES variant of type above-indicted namely those in which valine insecond position of NES is replaced with another hydrophobic amino acid(FIG. 6 and Table 3). Table 3 shows the correlation between NES motifand the tryptophans 288 and 290 in 40 NPM mutant proteins of leukaemicpatients.

TABLE 3 NPM NES Mutants Mut W Mut W Variant Motif (n=) (288) (290) 1L-XXX-V-XX-V-X-L 26/40  26/26  6/26 2 L-XXX-L-XX-V-X-L 6/40  1/6* 6/6 3L-XXX-F-XX-V-X-L 3/40 0/3 3/3 4 L-XXX-M-XX-V-X-L 2/40 0/2 2/2 5L-XXX-C-XX-V-X-L 2/40 0/2 2/2 6 L-XXX-F-XXX-L-FKKIV 1/40 0/1 1/1 *Themutation Q of FIG. 6 causes a mutation of both two tryptophans 288 and290 in presence of the variant 2 of NES (L-xxx-L-xx-V-x-L); mutW:mutated tryptophan.

The most common NES motif is the variant 1; the other NES variants (type2-6) are less frequent and they differentiate from the variant 1 for thepresence, in the place of Valine (V) in NES second position, of aLeucine (L), Phenylalanine (F), Methionine (M), or Cysteine (C).

Cytoplasmic Expression of NPM Mutants is a NES-Dependent Event

The fact that all NPM mutants contain a new NES motif in theircarboxyl-terminal portion suggests that the cytoplasmic removal of NPMcan result from an active transport of NPM mutants by means of Crm1, thereceptor delegated to the protein transport from nucleus to cytoplasm.

The authors have carried out some experiments to verify whether thenucleus-cytoplasm transport of NPM leukaemic mutants is altered somehow,in the presence of substances that, as already known, inhibit theactivity of the Crm1/Esportin 1, as well as Leptomicin B or theRatjadons.

The results of the experiments are clear. FIG. 7 shows as the nuclearexport of NPM mutants is NES-dependent. Under basal conditions, HI299 orNIH3T3 cells transfected with cDNAs that encode for labelled NPM mutantproteins show the expected aberrant cytoplasmic localization of mutants.In contrast, in the presence of Leptomicin B (LMB), NPM mutant proteinsare re-localized from the cytoplasmic to the nuclear compartment(nucleoplasm) (panels 7A and 7B). FIG. 7C-E shows the analysis atdifferent time points of the LMB effects on the mutant A associated toeGFP (eGFP-NPMmutA) in NIH-3T3 cells: the addition of LMB results in areduction of fluorescence in cytoplasm and Golgi area and concomitantfluorescence increase in nucleoplasm.

About 50% of mutant type A (the more common one) is re-localized innucleus in 20 minutes and the whole process is completed in 1 hour(panels C-D).

The Western-blot analysis of the sub-cellular distribution of Type A NPMmutant bound to GFP in Leptomicin B treated N1H-3T3 cells confirmedthat, over time, the GFP-NPM protein mutant A (molecular weight 64 kDa),unlike the GFP protein (molecular weight 27 kDa), progressivelyaccumulates in the pellet containing nucleuses (FIG. 7E). On thecontrary, in untreated NIH-3T3 cells, both GFP-NPM and GFP proteins are,as expected, only in the cytoplasmic fraction. In fact, the LMBtreatment induces a time-dependent accumulation of eGFP-NPMmutA in thepellet fractions (P). The purity of the sub-cellular fractions wasmeasured by removal of the antibody and blotting with an anti-β-tubulinantibody (inferior panel FIG. 7E). A not significant contamination wasmeasured for the over-expressed proteins (as it is clear in GFPblotting) (middle panel). In untreated cells, eGFP-NPMmutA was foundonly in the cytoplasmic fractions (C). The experiment was carried out inthe absence of dicycloeximide so that the continuous presence ofGFP-NPMmA in the cytoplasmic fractions during the treatment with LMB hasbeen shown over time.

The confocal microscope analysis of cells transfected with variousconstructs of NPM-EFG clearly shows that NPM mutants, after Leptomicin Btreatment, are re-localized in the nucleus and, specifically, innucleoplasm (FIG. 8A, on top), rather than in the nucleolus, that is theplace in which physiologically the wild-type NPM protein localizes (FIG.8A, middle). The nucleoplasmic re-localization of mutants by LeptomicinB is also shown through double staining at confocal microscope thatunderlines the presence of a mutual exclusiveness between thelocalization places of NPM mutant, displaced in nucleoplasm, andnucleolin (C23) that, as expected, is selectively expressed at nucleolarlevel (FIG. 8A, in bottom).

The nucleoplasmic re-localization of NPM after treatment with Crm1inhibitors was also confirmed on cells of patients with NPMc+ AML (FIG.8B).

An identical effect of the Crm1 inhibitors on the mutant was alsoobserved in OCI-AML3 human myeloid line that includes type A NPMmutation (FIG. 9A). In these cells, by co-precipitation experiments adirect physical interaction between mutated NPM protein and Crm1 wasalso shown (FIG. 9B).

The fundamental role played by NES motif in the process of expulsionfrom nucleus of NPM mutants and their consequent accumulation incytoplasm, is shown also by site-directed mutagenesis experiments. Infact, the substitution in type A NPM mutant of two valines of NES fortwo glycines (NPM mutA no-NES), vanishes the ability of mutant to beexported from nucleus to cytoplasm (FIG. 10).

Cytoplasmic Accumulation of Mutants Depends on the Coordinated Action ofNES and Mutations of Tryptophans 288 and 290

The role of two tryptophans 288 and 290 in NPM cytoplasmic accumulationwas evaluated using natural NPM mutants (from leukaemic patients) andNPM mutants constructed by site-directed mutagenesis. To assess theeffect on nucleolar bond of a single mutation at level of tryptophan290, we used the natural leukaemic mutant type E that, as illustrated inFIG. 6, maintains tryptophan 288. Following treatment with Leptomicin B,type E NPM mutant is re-localized at nuclear level. However, unlike thatobserved with mutant A, the re-localization occurs not only atnucleoplasma but also at nucleolus level (FIG. 10). A nuclearcompartment distribution very similar to that of mutant E, is alsoobserved with an artificial construct of type A NPM mutant in which themutated tryptophan in position 290 was re-inserted by site-directedmutagenesis (A290W). When both two tryptophans 288 and 290 arere-inserted in mutant A (C288W+A290W), the mutant protein, despite thepresence of NES, locates completely in nucleoli, independently from thepresence of Leptomicin B. The results of these experiments areillustrated in FIG. 10.

These observations clearly show that the tryptophans 288 and 290contribute significantly to NES-mediated nuclear expulsion of NPMmutants. In conclusion, in order that the aberrant cytoplasmicaccumulation of NPM occurs, it is necessary that NES and the mutationsof two tryptophans (or only tryptophan 290) act in combination. In otherwords, it is impossible to have cytoplasmic accumulation of NPM mutantswhen only NES is present in the absence of mutations of two tryptophans288 and 290 (or only tryptophan 290), or vice versa.

NPM Mutants Dislocate the NPM Wild-Type Protein from its PhysiologicalPlace (Nucleolus) to the Cytoplasm

Because all leukaemic NPM mutated proteins preserve the dimerizationdomain in the N-terminal site, it can be hypothesized that they can formheterodimers with NPM wild-type protein, like among the fusion proteins(NPM-ALK and NPM-MLF1) and same NPM wild-type protein.

FIG. 11A shows that, by heterodimerization mechanism, the mutants areable to bind and dislocate the NPM wild-type protein in cytoplasm. Infact, by co-transfecting H1299 cells with vectors encoding for(wild-type)-HA NPM and (mutant A)-eGFP NPM, it is observed that themutant and the wild-type NPM protein co-locate in cytoplasm. About 30%of cells were transfected and for about 70% of these, NPM mutant causesa partial recruitment of the wild-type form of NPM from the nucleoli tonucleoplasm and cytoplasm. These results are also confirmed byco-precipitation experiments of wild-type NPM (HA labelled) and mutantNPM (Flag labelled) (FIG. 11B). To transfect H1299 cells, plasmidsencoding for FH-PMwt and FH-NPM mutant A were used. In the left panel ofFIG. 11B, 5% of whole lysed cells derived from stably transfected cellswith FH-NPMwt, FH-NPM mutant A or H1299 control cells was subjected toWestern blot with α-NPM or α-HA. In the right panel, the 95% remainingof lysed cells was immuno-precipitated with an anti-Flag antibody (M2),and used for Western blot with α-NPM or it α-HA.

The possible mechanism of the altered nucleus-cytoplasm restrictedtransport of mutants and wild-type NPMs is schematised in FIG. 11C.

Immunohistochemistry to Predict all Mutations at Level of Exon 12 of NPMGene

As above illustrated, the mechanism responsible of the accumulation ofNPM mutant proteins in the cytoplasm of leukaemic cells, depend on themutations of tryptophans 288 and 290 and creation of NES. Because thesealterations are present in all till now identified leukaemic NPMmutants, it is hypothesized that the immunohistochemical staining withanti-NPM antibodies is able to predict, by demonstrating cytoplasmicdelocalization of NPM, all mutations occurring at level of exon 12 ofNPM gene.

To verify this hypothesis, we have compared the sub-cellular expressionof NPM protein (nuclear vs cytoplasmic) with the mutational state of NPMgene. The study was carried out on 393 patients with AML of GIMEMAAML99P/AML 12 EORTCs protocol. Obtained results clearly show that thepresence of a cytoplasmic positivity for NPM is predictive with absolutespecificity of mutations at level of exon 12 of NPM (Table 4).

TABLE 4 NPM gene mutations LAM (N = 373) NPM protein localization*(Exon-12) 191 Cytoplasmic 191/191 202 Nuclear  0/202 *Determined onparaffin embedded sections with monoclonal anti-NPM antibodies

Immunohistochemical test can be used for diagnostic purpose as indicatedin FIG. 12. The test is rapid, economic, easily interpretable, highlysensitive and specific. For all these reasons it could be used as firststep in the molecular characterization of AMLs. In fact in NPMc+ AMLs itisn't necessary to carry out cytogenetic, FISH and molecular analysis,for the major chromosomal alterations, such as t(15;17), t(8;21), inv16,t(6;9) and 11q23/MLL because they are mutually exclusive with thecytoplasmic positivity for NPM. On the contrary, in NPMc AMLs, theseanalyses are compulsory. Cytogenetics helps for the identification ofrare translocations with potential prognostic impact in 14% of NPMc+ AMLwith minor chromosomal anomalies. Mutations of FLT3 gene should besearched in all AML patients AML (independently on NPM) because itscorrelation with the sub-cellular expression of NPM can help to identifynew prognostic subgroups in normal karyotype AML (Schnittger et al.,2005; Dohner et al., 2005; Verhaak et al., 2005). The use ofimmunohistochemistry to identify NPM mutations has also a clinicalimportance, because the cytoplasmic distribution of NPM and genemutations are predictive of a good response to induction therapy, and abetter long-term prognosis compared with cases of acute leukaemia withnormal karyotype without mutation of NPM gene (NPMc-AML) (Schnittger etal., 2005; Dohner et al, 2005; Verhaak et al., 2005).

The above illustrated data explain mechanism through which the exon 12specific mutations of NPM gene alter the nucleus-cytoplasm transport ofmutated and wild-type NPM proteins. The mechanism is identical both intransfected and leukaemic cells of patients with NPMc+ AML and inOCI/AML3 human leukaemic line. Particularly, the mutations lead to twofundamental changes in the carboxyl-terminated region of NPM mutants: 1)a NES is produced that potentiates the expulsion of mutant proteins byCrm1; and 2) two tryptophans 288 and 290 (or only tryptophan 290) arelost which, under normal conditions, are essential for the bond of NPMprotein to nucleoli.

The primary sequence analysis of wild-type NPM protein allowed to detectan hypothetical physiological NES of LxxPxxLxL type, which is localizedin the zone between residues 94 and 102 of NPM (Wang et al., 2005).Despite the presence of this NES, the wild-type NPM protein, underphysiological conditions, locates mainly in nucleoli and this suggeststhat the part of NPM protein that is normally expelled from nucleus tocytoplasm through physiological NES, is decidedly inferior to that ofthe same protein that, by two NLSs (“nuclear localization signals”), isable to go back from cytoplasm into nucleus. Because the murine andhuman artificial wild-type NPM mutants without the two tryptophans 288and 290 (different with respect to the leukaemic Type A NPM mutant onlyfor the lack of C-terminal NES) locate exclusively in nucleoplasm(Nishimura et al., 2002), it is very likely that the additional NES,created by the mutation at level of C-terminal region, confers toleukaemic NPM mutant a greater ability to be exported out of nucleus;this could be due to the additive effect and/or increased Crm1 affinityof the second NES.

Although both, 288 and 290, play a role in the nucleolar localization ofNPM, tryptophan 290 could be more important, because it is constantlyaltered in all till now identified leukaemic NPM mutants. The mutationof both two tryptophans allows the maximum inhibition of the nucleolarbond and nucleoplasmic delocalization of mutants to be achieved inleukaemic NPMc+. cell Of great importance is the observation that NESmotif more commonly found in NPM mutants (LxxxVxxVxL) is alwaysassociated with mutations of both tryptophans. In contrast, tryptophan288 appears to be maintained only in those NPM mutants including lesscommon variants of NES, namely those characterized by the presence ofleucine phenylalanine, cysteine or methionine in the second position ofNES, in place of valine (Table 3). These two observations indicate alikely functional difference among NESs of the C-terminal region ofleukaemic NPM mutants.

The results of our studies show also unequivocally that the aberrantcytoplasmic accumulation of mutants can occur only due to thecoordinated action of NES and mutated tryptophans. It is possible thatthe anomalous accumulation of NPM mutants occurs according to thefollowing mechanism: i) mutated leukaemic NPM proteins preserving twonuclear localization signals (NLS), enter into nucleus; ii) theirability to bind the nucleoli is completely inhibited when thetryptophans are mutated, or partially inhibited when only tryptophan 290is altered, resulting in the accumulation of mutants in nucleoplasm;iii) nucleoplasmic mutants are caught by Crm1 that determines rapidexpulsion thereof in cytoplasm where they progressively accumulate.

The explanation of the altered transport mechanism of NPM in NPMc+leukaemia suggests, as possible therapeutic intervention area, the“re-localization” of leukaemic NPM mutants and wild-type NPM protein intheir physiological sites, through the use of Crm1 inhibitors or smallsynthesis molecules that interfere with the NPM mutant-Crm1 bond orwild-type NPM protein or other molecules able to interact with NPM (ARF,etc.).

EXAMPLE 5 Development of a Quantitative POR System for Evaluation andMonitoring of Minimal Residual Disease

Several heterozygote NPM1 mutations suggest the necessity of amutation-specific system for disease monitoring. In the development ofsystem it is worth considering that the two most frequent mutations,so-called mutation A and B, include over 95% of all mutated cases.

Materials and Methods

A specific evaluation method uses a forward primer designed on exon 11(cNPM1-F: 5′-5′-GAAGAATTGCTTCCGGATGACT-3′), a probe on the junction exon11/exon 12 (c.Probe: 5′-FAM-ACCAAGAGGCTATTCAA-MGB-3′) andmutation-specific reverse primers (cNPM mut.A-R:5′-CTTCCTCCACTGCCAGACAGA-3′ and cNPM mut.B-R:5′-TTCCTCCACTGCCATGCAG-3′). Forward primer and probe are the sameregardless of different mutations (FIG. 13).

Step 1=Retro-transcription reaction according to EAC protocol (Gabert etal., Leukaemia 2003).

Step 2=Amplification reaction uses a mixture containing 12.5 μl of TaqMan universal PCR Master Mix (Applied Biosystem), 300 nM Primers, 200 nMof probe and 5 μl of cDNA in a total volume of 25 μl. Conditions: 2minutes at 50° C. (UNG enzyme activation), 10 min at 95° C. (UNG enzymeinactivation and AmpliTaq polymerase activation) followed by 50 cyclesat 95° C. for 15 seconds, at 62° C. for 1 minute for mutation A and at59° C. for 1 minute for mutation B. As quantitative and qualitative RNAcontrol ABL gene can be amplified. The analysis setting of instrument(ABI PRISM 7700 Sequence Detection System, Applied Biosystem) includes a“threshold” of 0.1 with “a baseline” from 3 to 15 both for ABL and NPM.System sensitivity and specificity are analyzed using sequentialdilutions, by a factor equal to 10, by mixing RNA extracted frommedullary leukaemic cells with NPM mutation type A or B and RNA obtainedfrom a pool of medullary cells from patients without NPM mutations(verified by sequencing).

The standard plot of absolute quantitative evaluation for mutation A isconstructed using a plasmid construct. Such a construct consists ofplasmid vector pCRII-TOPO, (Invitrogen, Groningen, Netherlands) plus aportion of NPM1 gene containing mutation A. The amplification ofmutation A is obtained by RT-PCR with the primers NPM1_(—)390_F(5′-GTCTTAAGGTTGAAGTGTGGT-3′) and NPM1_(—)1043_R(5′-TCAACTGTTACAGAAATGAAATAAGACG-3′).

The plasmid is prepared in five sequential dilutions: 10⁵, 10⁴, 10³,10², 10 copies. The results of RQ-PCR for mutation A normalized on ABLtranscripts are expressed as copy number of NPM with mutation A every10⁴ copies of ABL.

The “maximum reproducible sensitivity”, according to guidelines aboutMinimal Residual Disease defined by an European Study Group (van derVelden V H J et al, 2003), is defined as the lowest dilution in whichall sample replicates are positive within a Ct (Cycle threshold) of 1.5,and the highest Ct of replicates is at least of 3.0 Ct lower than thelowest value of background. The “maximum sensitivity” is defined asminimum dilution in which at least a sample is positive and at least 1.0Ct lower than the smallest Ct of background. With these definitions, aresult is defined as “positive, not quantifiable” in the presence ofamplified in 1 of 2 replicates below the maximum reproduciblesensitivity, but still 1.0 Ct lower than the lowest background.

Results

The sensitivity and specificity of “Reverse Quantitative” PCR (RQ-PCR)were tested in the dilution series of 10⁵, 10⁴, 10³, 10², 10 plasmidcopies. The Ct values (Cycle Threshold) and the “slope” of plot forplasmid are shown in the following figure.

The standard plasmid plots show a “mean slope” of −0.38 and an“intercept” of 39.5±0.45 Ct. The correlation coefficient is high (>0.99in all experiments) and demonstrates the accurate identification of thepresence of copies of mutation A in unknown samples. The maximumreproducible sensitivity of RQ-PCR corresponds to 10 plasmid molecules(FIG. 14).

Sequential dilutions based on a factor equal to 10 were carried out tosimulate the sensitivity and reproducibility of RQ-PCR in monitoring ofminimal residual disease in 5 patient diagnosed as afflicted with (acutemyeloblastic leukaemia) NPMc+ AML (4 patients with mutation A and 1 withmutation B).

RNA extracted from medullary leukaemic cells with type A or B NPMmutation was diluted with RNA achieved from a pool of medullary cellsfrom patients without NPM mutations.

Maximum reproducible sensitivity equal to 10⁻⁴ was found in all 5patients with mutations A or B, while the maximum sensitivity was 10⁻⁶in the sample with type B mutation and in 3 of 4 samples with type Amutation. The maximum sensitivity equal to 10⁻⁵ was observed in one ofsamples with A mutation. The background amplification was observed onlyin one case and at very high Ct values (Ct>48), showing the highspecificity of system (FIG. 15).

Using the plasmidic calibration plot 13 patients afflicted with AML withNPM mutation type A were analyzed at diagnosis and after inductiontreatment.

The results were expressed as “copy number of NPM1 Mut.A/10000 copies ofABL.” At diagnosis, all samples show >30000 copies. After inductiontreatment the copy number diminishes markedly in 10 patients thatevidenced complete haematological remission. In 5 patients the reachedcopy number was <70 while in the other 5 patients it ranged between 580and 5046. A small or no diminution of the copy number was evidenced in 3cases: 2 complete remissions and 1 partial remission (FIG. 16).

In 3 patient afflicted with AML with mutations of type A NPM1 gene theaforesaid system was used for monitoring the minimal residual diseaseboth during the therapy and in the follow-up.

The cDNA specific RQ-PCR showed a copy number <10 after the first or thesecond cycle of consolidation therapy. A different kinetics in thediminution of the copy number was observed in the three samples as shownin FIG. 17. A small but persistent number of mutated copies isassociated with the haematological remission in a patient (square). Inone of the remaining cases the number of mutated copies markedlydecreases after consolidation treatment (diamonds); such diminution isless pronounced in the second patient (triangles). In the last twopatients the copy number again increases and in both caseshaematological relapse occurs (FIG. 17).

In conclusion the system has such sensitivity, specificity andreproducibility characteristics to be used in clinical tests.

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1.-71. (canceled)
 72. Oligonucleotide sequences encoding for mutatednucleophosmin protein (NPM) characterized by having a cytoplasmiclocation and comprising mutated amino acid sequence wherein one of thetryptophan residues 288 and/or 290, and/or a signal motif of nuclearexport (NES) are present in the C-terminal region of the sequence of thehuman nucleophosmin (NP-002511), and wherein said sequences aredeoxyribonucleotide or ribonucleotide sequences or complementarysequences thereof.
 73. Oligonucleotide sequences encoding for proteinaccording to claim 72, wherein both tryptophan residues 288 and 290 aremutated.
 74. Oligonucleotide sequences encoding for protein according toclaim 72, wherein only the tryptophan residue 290 is mutated. 75.Oligonucleotide sequences encoding for protein according to any one ofclaims from 72 to 74, wherein said nuclear export signal motif (NES)comprises an amino acidic sequence YxxxYxxYxY wherein Y is a hydrophobicamino acid selected from the group consisting of leucine, isoleucine,methionine, valine, phenylalanine, and x can 15 be any amino acid orfragments and variants thereof; and wherein the sequence YxxxYxxYxY isselected from the group consisting of LxxxVxxVxL (SEQ ID No I),LxxxLxxVxL (SEQ ID No 2), LxxxFxxVxL (SEQ ID No 3), LxxxMxxVxL (SEQ IDNo 4), LxxxCxxVxL (SEQ ID No 5).
 76. Oligonucleotide sequences encodingfor proteins according to claim 75 wherein LxxxVxxVxL (SEQ ID No 1) isselected from the group consisting of LCLAVEEVSL (SEQ ID No 6);LCMAVEEVSL (SEQ ID No 7); LCVAVEEVSL (SEQ ID No 8); LSRAVEEVSL (SEQ IDNo 9); LCTAVEEVSL (SEQ ID No 10); LSQAVEEVSL (SEQ ID No 10); LCHAVEEVSL(SEQ ID No 12); LCRAVEEVSL (SEQ ID No 13); LCRGVEEVSL (SEQ ID No 14);LCQAVEEVSL (SEQ ID No 15); LCAAVEEVSL (SEQ ID No 16); LCKAVEEVSL (SEQ IDNo 17); LxxxLxxVxL (SEQ ID No 2) is selected from the group consistingof LWQSLAQVSL (SEQ ID No 18); LWQSLEKVSL (SEQ ID No 19); LWQSLSKVSL (SEQID No 20); LCTFLEEVSL (SEQ ID No 21); LxxxFxxVxL (SEQ ID No 3) isselected from the group consisting of LWQCFAQVSL (SEQ ID No 22);LWQCFSKVSL (SEQ ID No 23); LWQRFQEVSL (SEQ ID No 24); LWQDFLNRL (SEQ IDNo 25); LxxxMxxVxL (SEQ ID No 4) is LWQSMEEVSL (SEQ ID No 26) orLWQRMEEVSL (SEQ ID No 27); LxxxCxxVxL (SEQ ID No 5) is LWQCCSQVSL (SEQID No 28).
 77. Oligonucleotide sequences encoding for protein accordingto claim 76 wherein said C-terminal region includes VSLRK peptide (SEQID No 29).
 78. Oligonucleotide sequences encoding for protein accordingto claim 77 further comprising a D-amino acid upstream of L-amino acidat the N-terminal end of the NES motif.
 79. Oligonucleotide sequencesencoding for protein according to claim 77 further characterized in thatit is fused to a reporter protein wherein said reporter protein isselected from the group consisting of EGFP, beta-galactosidase,luciferase, GFP.
 80. Oligonucleotide sequences according to claim 77wherein said deoxyribonucleotide sequences include at least one of thesequences having the followings GenBank deposit numbers: AY740634,AY740635, AY740636, AY740637 AY740638, AY740639.
 81. Oligonucleotidesequences according to claim 77 wherein said sequences are labeled withan agent selected from the group consisting of fluorescent substance,biotin, radioisotope, nanoparticle.
 82. The method of diagnosing acutemyeloid leukemia and monitoring residual disease by detecting NPMmutations with at least one of the primers and/or oligonucleotide probescorresponding to sequences as defined in claims 72-79.
 83. The methodaccording to claim 82, wherein said primer or oligonucleotide probe islabeled with an agent selected from the group consisting of fluorescentsubstance, biotin, radioisotope, nanoparticle.
 84. The method accordingto claim 82 further comprising the primers with the followingoligonucleotide sequences: i) NPM1_25F: (SEQ ID No 36)5′-GGTTGTTCTCTGGAGCAGCGTTC-3′; NPM1_111 2R: (SEQ ID No 37)5′-CCTGGACAACATTTATCAAACACGGTA-3′; ii) NPM1_390F: (SEQ ID No 38)5′-GGTCTTAAGGTTGAAGTGTGGT-3′; NPM1_1043_R; (SEQ ID No 39)5′-TCAACTGTTACAGAAATGAAATAAGACG-3′; iii) NPM_940F_mutA (SEQ ID No 40)5′-GAGGCTATTCAAGATCTCTGTCT-3′; NPM1_1112 R (SEQ ID No 41)5′-CCTGGACAACATTTATCAAAGACGGTA-3′; iv) NPM_940F_mutB (SEQ ID No 42)5′-GAGGCTATTCAAGATCTCTGCAT 3′; NPM1_1112 R (SEQ ID No 43)5′-CCTGGACAACATTTATCAAACACGGTA3′; v) NPM_940F_mutC (SEQ ID No 44)5′-GAGGCTATTCAAGATCTCTGCGT-3′; NPM1_1112 R (SEQ ID No 45)5′-CCTGGACAACATTTATCAAACACGGTA-3′; vi) NPM_940F_mutD (SEQ ID No 46)5′-GAGGCTATTCMGATCTCTGCCT-3′; NPM1_1112 R (SEQ ID No 47)5′-CGTGGACAACATTTATCAAACACGGTA-3′; vii) NPM1 -F: (SEQ ID No 48)5′-TTAACTCTCTGGTGGTAGAATGAA-3′; NPM1-R: (SEQ ID No 49)5′-CCAGACTATTTGCCATTCCTAAC-3′; viii) NPM1_89F_BamHI: (SEQ ID No 50)5′-GCCACGGATCCGAAGATTCGATGGAC-3′; NPM1_1044R_EcoRI: (SEQ ID No 51)5′ATCAAGAATTCCAGAAATGAAATAAGA CG-3′; ix) cNPM-F: (SEQ ID No 52)5′-GAAGAATTGCTTCCGGATGACT-3′ and cNPM mut.A-R: (SEQ ID No 53)5′-CTTCCTCCACTGCCAGACAGA-3′ or cNPM mut.B-R: (SEQ ID No 54)5′-TTCCTCCACTGCCATGCAG-3′.


85. Expression vector comprising oligonucleotide sequences as defined inclaim
 72. 86. Host cell permissive for at least one of mutated proteinsas defined in claim
 72. 87. The method of employing the host cellaccording to claim 86 as study model of NPMc+ AML for the screening ofnew drugs.